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| Status |
Public on Apr 16, 2019 |
| Title |
mPouV-Sox3-LOF_exp4_st10p_polyA-RNA_rep3 |
| Sample type |
SRA |
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| Source name |
whole embryo
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| Organism |
Xenopus tropicalis |
| Characteristics |
strain: wild-type (out-bred Nigerian)
developmental stage: 10+ (early gastrula)
treatment: injection of mPouV/Sox3 MOs
chip antibody: none
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| Treatment protocol |
Injections were carried out into the zygote's animal pole: MyoD-HA mRNA (80 pg); mPouV MO (5 ng Pou5f3.2 MO, 5 ng Pou5f3.3 MO and 5 ng standard control MO); mPouV/Sox3 MOs (5 ng Pou5f3.2 MO, 5 ng Pou5f3.3 MO and 5 ng Sox3 MO); β-catenin MO (5 ng); mVegT MO (10 ng); standard control MO (5-10 ng according to the dose used for the β-catenin or mVegT loss-of-function experiment); and α-amanitin (30 pg). Embryos were treated with 100 µM SB431542 or 10 µM LDN19318 from the 8-cell stage onwards to block Nodal or BMP signaling. Control embryos were treated accordingly with DMSO, the solvent of these antagonists.
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| Growth protocol |
Embryos were grown to the indicated developmental stage in 5% MMR at 25ºC.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Chromatin immunoprecipitation (ChIP): Briefly, dejellied X. tropicalis embryos were treated with 1% formaldehyde (in 1% Marc’s Modified Ringer’s, MMR) for 15-45 min at room temperature to cross-link chromatin proteins to nearby genomic DNA. Duration of fixation was determined empirically and depended mainly on the developmental stage and antibody epitopes as described in Gentsch and Smith (2017). Efficient Preparation of High-Complexity ChIP-Seq Profiles from Early Xenopus Embryos. Methods in Molecular Biology, 1507, 23-42. Fixation was terminated by rinsing embryos three times with ice-cold 1% MMR. If required, post-fixation embryos were dissected to select specific anatomical regions in ice-cold 1% MMR. Fixed embryos were homogenized in CEWB1 (10 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.1% SDS) supplemented with 0.5 mM DTT, protease inhibitors and, if using phospho-specific antibodies, phosphatase blockers (0.5 mM orthovanadate and 2.5 mM NaF). To solubilise yolk platelets and separate them from the nuclei, the homogenate was left on ice for 5 min and then centrifuged (1000 g) for 5 min at 4ºC. Homogenization and centrifugation was repeated once before resuspending the nuclei containing pellet in 1-3 ml CEWB1. Nuclear chromatin was solubilized and fragmented by isothermal focused or microtip-mediated sonication. The solution of fragmented chromatin was cleared by centrifuging (15,000 g) for 5 min at 4ºC. If required, ~1% of the cleared chromatin extract was set aside for the input sample (negative control for ChIP). ChIP-grade antibodies were used to recognize specific chromatin features and to enrich these by coupling the antibody-chromatin complex to protein G magnetic beads and extensive washing. These steps were carried out at 4ºC. The beads were washed twice in CEWB1, twice in WB2 (10 mM Tris pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.1% SDS), twice in WB3 (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Igepal CA-630 and 1% sodium deoxycholate) and once in TEN (10 mM Tris pH 8.0, 150 mM NaCl and 1 mM EDTA). ChIP was eluted off the beads twice with 100 µl SDS elution buffer (50 mM Tris pH 8.0, 1 mM EDTA and 1% SDS) at 65ºC. ChIP eluates were pooled before reversing DNA-protein cross-links. Input (filled up to 200 µl with SDS elution buffer) and ChIP samples were supplemented with 10 µl 5 M NaCl and incubated for 6-16 h at 65ºC in a hybridization oven. Samples were treated with proteinase K and RNase to remove any proteins and RNA from the co-immunoprecipitated DNA fragments. The DNA was purified with phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.9) using phase-lock gel heavy microcentrifuge tubes for phase separation and precipitated with 1/70 volume of 5 M NaCl, 2 volumes of absolute ethanol and 15 µg blue glycogen (GlycoBlue). After centrifugation, the DNA pellet was air-dried and dissolved in 11 µl elution buffer (10 mM Tris-HCl, pH 8.5). The DNA concentration was determined on a fluorometer using high-sensitivity reagents for double-stranded DNA (10 pg/µl to 100 ng/µl).
DNase-probed chromatin accessibility: The assay to probe chromatin accessibility with DNase was adapted to early X. tropicalis embryos using a novel approach. Ultracentrifugation- or gel electrophoresis-mediated size selection was replaced by two rounds of solid phase reverse immobilization (SPRI) to remove high molecular weight (HMW) DNA from the informative, short DNA fragments. Wide-bore pipette tips were used for the resuspensions and the transfers of biological samples from the second homogenization step until after SPRI to avoid the shearing of HMW DNA. About 250 dejellied mid-blastula embryos (stage 8+) were collected in 2-ml round-bottom microcentrifuge tubes and homogenized in 2 ml ice-cold LB-DNase buffer (15 mM Tris-HCl pH 8.0, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM and 0.5 mM spermidine) supplemented with 0.05% Igepal CA-630. The homogenate was left on ice for 3 min before centrifuging (1,000 g) for 2 min at 4ºC. The pellet was gently resuspended in 2 ml ice-cold LB-DNase buffer (without Igepal CA-630) before centrifuging (1,000 g) again for 2 min at 4ºC. The pellet was resuspended in 600 µl of room temperature LB-DNase buffer supplemented with 6 mM CaCl2. The sample was distributed equally to two 1.5-ml microcentrifuge tubes. Approximately 0.1 U DNase I was added to one aliquot while leaving the other aliquot untreated (internal control). Both samples were incubated for 8 min at 37ºC before adding 300 µl STOP buffer (50 mM Tris pH8.0, 100 mM NaCl, 100 mM EDTA, 0.1% SDS, 80 µg RNase A, 333 nM spermine and 1 µM spermidine). The samples were incubated for 15 min at 55ºC. Next, they were digested with 200 µg proteinase K for 2 h at 55ºC. The digests were transferred to phase-lock gel heavy microcentrifuge tubes for phenol:chloroform:isoamylalcohol (25:24:1) purification. The DNA was precipitated with 1/20 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of absolute ethanol. The DNA pellets were dissolved in 27 µl elution buffer (10 mM Tris pH8.5). To remove any remaining RNA (i.e. not digested in a first round of RNase treatment), 10 µg RNase A were added to the DNA samples. 5 µl (an equivalent of 20 mid-blastula embryos) from the internal control was digested with 0.3 U DNase I for 5 min at 37ºC to generate a negative control profile (i.e. DNase-treated naked genomic DNA) for chromatin accessibility. The purification and precipitation of negative control DNA was carried out along with the chromatin digest after SPRI-mediated size selection. The DNA sample from the chromatin digest was further processed by two rounds of SPRI. 22.5 µl SPRI beads were added to 25 µl DNA sample without pipetting up and down. After 3 min, by which time HMW DNA caused beads to coalesce, the tubes were clipped into a magnetic stand for microcentrifuge tubes. After 3 min, the supernatant was transferred to a 96-well microplate. 47.5 µl elution buffer and 43 µl SPRI beads were added sequentially and mixed gently by slowly pipetting up and down. After 3 min, the plate was transferred to a magnetic stand for 96-well plates. Once the beads have settled to bottom of the well, the supernatant and the digest of naked genomic DNA (see above) were transferred to separate 1.5-ml phase-lock gel heavy microcentrifuge tubes and purified with phenol:chloroform:isoamylalcohol (25:24:1, pH7.9). The DNA fragments were precipitated with 1/20 volume of 3 M sodium acetate (pH 5.2) and 3 volumes of absolute ethanol. After centrifugation, the DNA pellets were dissolved in 12 µl elution buffer. The DNA concentrations were determined on a fluorometer using high-sensitivity reagents for double-stranded DNA (10 pg/µl to 100 ng/µl).
Chromatin conformation capture (3C): About 500 dejellied mid-blastula embryos (stage 8+) were fixed with 1% formaldehyde (in 1% Marc’s Modified Ringer’s, MMR) for 40 min at room temperature. The fixation reaction was terminated by rinsing the embryos three times with ice-cold 1% MMR. The embryos were aliquoted equally into two 2-ml round-bottom microcentrifuge tubes and homogenized in 2 ml ice-cold CEB-3C (10 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.2% SDS) supplemented with protease inhibitors and 0.05 mM DTT (=CEB-3C*). The homogenates were kept on ice for 5 min before centrifuging (1,000 g) for 2 min at 4ºC. The pellet was resuspended in 0.5 ml ice-cold CEB-3C*. The resuspensions were pooled and then equally divided to two 50-ml conical tubes and each filled up with CEB-3C to 50 ml. The embryonic extracts were incubated at 37ºC for 1 h in a hybridization oven with the tubes rotating inside a hybridization bottle. The tubes were then centrifuged (1,000 g) for 5 min at room temperature. The pellets were resuspended in 50 ml double distilled water. The tubes were centrifuged (1,000 g) agai
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
mPouV/Sox3 loss-of-function loss-of-function, stage 10+ (early gastrula), experiment 4, poly(A)+ RNA, biological replicate 3
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| Data processing |
Bases were called using Casava version 1.8.2
ChIP-Seq: Single reads of maximal 50 bases were processed using trim_galore v0.4.2 to trim off low-quality bases and adapter contamination from the 3’ end. Processed reads were aligned to the X. tropicalis genome assembly v7.1 (and v9.1 for some ChIP-Seq) running Bowtie2 v2.2.9 with default settings. Alignments were converted to HOMER’s tag density format with redundant reads being removed (makeTagDirectory -single -tbp 1 -unique -mapq 10 -fragLength 175 -totalReads all). For all genomic regions except for the mir427 gene cluster, only uniquely aligned reads (i.e. MAPQ ≥10) were processed. The highly repetitive mir427 locus was profiled with all reads only best aligning up to 80 times (-k 80) to this region. In this case the mass of each read was normalised to the number of alignments. We pooled all input alignments from various developmental stages. This created a comprehensive mappability profile that covered ~400 million unique base pair positions. All chromatin profiles were position-adjusted and normalised to the effective total of 1 million aligned reads including multimappers (counts per million aligned reads, CPM). Transcription factor binding sites were identified using the following or otherwise default parameters of HOMER’s calling of ChIP-mediated read enrichments (hereafter called peaks): findpeaks -style factor -minDist 175 -fragLength 175 -inputFragLength 175 -fdr 0.001 -gsize 1.435e9 -F 3 -L 1 -C 0.97. This means that both ChIP and input alignments were extended 3’ to 175 bp for the detection of significant (0.1% FDR) peaks being separated by ≥175 bp. The effective size of the X. tropicalis genome assembly v7.1 was set to 1.435 billion bp, an estimate obtained from the mappability profile. These peaks showed equal or higher tag density than the surrounding 10 kb, ≥3-fold more tags than the input and ≥0.97 unique tag positions relative to the expected number of tags. To detect focal RNAPII recruitment to putative cis-regulatory elements and avoid calling peaks within broad regions of RNAPII elongation, the threshold of focal ratio and local enrichment within 10 kb was elevated to 0.6 and 3 (-L 3), respectively. To further eliminate any false positive peaks, we removed any peaks with <0.5 (transcription factors including signal mediators) or <1 (RNAPII) CPM and those falling into blacklisted regions showing equivocal mappability due to genome assembly errors, gaps or simple/tandem repeats. Regions of equivocal mappability were identified by a 2-fold lower (poor) or 3-fold higher (excessive) read coverage than the average detected in 400-bp windows sliding at 200-bp intervals through normalised ChIP input and DNase-digested naked genomic DNA. All identified regions ≤800 bp apart were subsequently merged. Gap coordinates were obtained from the Francis Crick UCSC genome browser (http://genomes.crick.ac.uk). Simple repeats were masked with RepeatMasker v4.0.6 using the crossmatch search engine v1.090518 and the following settings: RepeatMasker -species "xenopus silurana tropicalis" -s -xsmall. Tandem repeats were masked with Kent’s trfBig wrapper script of the Tandem Repeat Finder v4.09 using the following settings: weight for match, 2; weight for mismatch, 7; delta, 7; matching probability, 80; indel probability, 10; minimal alignment score, 50; maximum period size, 2,000; and longest tandem repeat array (-l), 2 [million bp].
DNase-Seq: Single and paired-end reads of maximal 50 bases were processed using trim_galore v0.4.2 to trim off low-quality bases and adapter contamination from the 3’ end. Processed reads were aligned to the X. tropicalis genome v7.1 and v9.1 using Bowtie2 v2.2.9 with default settings apart from -X (fragment length), which was reduced to 250 bp for paired-end reads. Alignments were sorted by genomic coordinates and only those with a quality score of ≥10 were retained using samtools v1.3.1. Duplicates were removed using Picard (MarkDuplicates). Paired-end alignments were dissociated using hex flags (-f 0x40 or 0x80) of samtools view. Single alignments were converted to HOMER’s tag density format (makeTagDirectory -single -unique -fragLength 100 -totalReads all). DNase hypersensitive sites (DHSs) were identified using the following or otherwise default parameters of HOMER’s peak calling: findpeaks -style factor -minDist 100 -fragLength 100 -inputFragLength 100 -fdr 0.001 -gsize 1.435e9 -F 3 -L 1 -C 0.97. This means that alignments of DNase-digested chromatin fragments (or naked genomic DNA fragments considered here as ‘input’) were extended 3’ by 100 bp from the DNase cleavage site to detect significant (0.1% FDR) DNase-mediated read enrichments (hereafter called peaks) being separated by ≥100 bp. The effective size of the X. tropicalis genome assembly v7.1 was set to 1.435 billion bp, an estimate obtained from the mappability profile (see ‘ChIP-Seq’ section). These peaks showed equal or higher tag density than the surrounding 10 kb, ≥3-fold more tags than the input and ≥0.97 unique tag positions relative to the expected number of tags. Peaks falling into blacklisted regions (see ‘ChIP-Seq’ section) were removed. DNase-probed chromatin accessibility profiles were position-adjusted and normalised to the effective total of 1 million aligned reads including multimappers.
Next-generation capture-C: Paired-end reads were processed using trim_galore v0.4.2 to trim off low-quality bases and adapter contamination from the 3’ end. Only complete mate pairs were processed further to reconstruct single reads from overlapping paired-end sequences using FLASH v1.2.11 with interleaved output settings for non-extended reads (flash --interleaved-output --max-overlap 150). FLASH reads were split in silico at DpnII restriction sites using a designated perl script (dpnII2E.pl, https://github.com/Hughes-Genome-Group/captureC/releases) before aligning them to the X. tropicalis genome assembly 7.1 using Bowtie2 v2.2.9. The alignment was run with default settings and one thread only to maintain the order of reads. The view function of samtools v1.3.1 was used to retain alignments with a quality score of ≥10. The alignments were analysed further using a suite of perl scripts (https://github.com/Hughes-Genome-Group/captureC/releases) modified to process both chromosome and scaffold coordinates. Viewpoint coordinates included a 1-kb proximity exclusion range. Restriction fragments were classified as capture, proximity-exclusion or reporter. PCR duplicates were removed. The interaction map was based on the number of unique paired-end reads per restriction fragment. Windows of 2 kb incrementing by 200 bp were used to consolidate interactions, which were normalised to 10,000 interactions per viewpoint. Significant chromatin conformation changes were identified using the R package DESeq2 v1.14.1.
RNase-Seq: Paired-end reads were aligned to the X. tropicalis genome assembly v7.1 using STAR v2.5.3a with default settings and a revised version of gene models v7.2 to improve mapping accuracy across splice junctions. The alignments were sorted by read name using the sort function of samtools v1.3.1. Exon and intron counts (-t 'exon;intron') were extracted from unstranded (-s 0) alignment files using VERSE v0.1.5 in featureCounts (default) mode (-z 0). Intron coordinates were adjusted to exclude any overlap with exon annotation. Differential expression analysis was performed with both raw exon and intron counts excluding those belonging to ribosomal and mitochondrial RNA using the R package DESeq2 v1.14.1. In an effort to find genes with consistent fold changes over time, p-values were generated according to a likelihood ratio test reflecting the probability of rejecting the reduced (~ developmental stage) over the full (~ developmental stage + condition) model. Resulting p-values were adjusted to obtain false discovery rates (FDR) according to the Benjamini-H
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| Submission date |
Apr 16, 2018 |
| Last update date |
Apr 16, 2019 |
| Contact name |
George E. Gentsch |
| E-mail(s) |
george.gentsch@crick.ac.uk
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| Organization name |
The Francis Crick Institute
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| Department |
Developmental Biology Laboratory
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| Lab |
James C. Smith
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| Street address |
1 Midland Road
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| City |
London |
| State/province |
Greater London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
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| Platform ID |
GPL23182 |
| Series (1) |
| GSE113186 |
Maternal pluripotency factors initiate extensive chromatin remodelling to predefine first response to inductive signals |
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| Relations |
| BioSample |
SAMN08937548 |
| SRA |
SRX3942725 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3099617_mPSkd_st10p_frogE_GG170.exon.txt.gz |
109.3 Kb |
(ftp)(http) |
TXT |
| GSM3099617_mPSkd_st10p_frogE_GG170.intron.txt.gz |
83.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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