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Status |
Public on Nov 21, 2020 |
Title |
Essrb-ChIP-nexus |
Sample type |
SRA |
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Source name |
Esrrb
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Organism |
Mus musculus |
Characteristics |
cell line: R1 ESCs cell type: embryonic stem cells chip antibody: anti-Esrrb (Abcam, ab19331)
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Growth protocol |
Mouse R1 ESCs were cultured on 0.1% gelatin-coated plates without feeder cells. Mouse ESC medium was prepared by supplementing N2B27 medium (1:1 mix of DMEM/F12 with GlutaMax supplemented with N2 and Neurobasal medium supplemented with B27, Invitrogen) with 2 mM L-Glutamine (Stemcell Technologies), 1x 2‑Mercaptoethanol (Millipore), 1x NEAA (Stemcell Technologies), 3 µM CHIR99021 (Stemcell Technologies), 1 µM PD0325901 (Stemcell Technologies), 0.033% BSA solution (Invitrogen) and 107 U/ml LIF (Millipore).
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Extracted molecule |
genomic DNA |
Extraction protocol |
For each ChIP experiment, 107 mouse ESCs were used. Cells were washed with PBS and cross-linked with 1% formaldehyde (Fisher Scientific) in PBS for 10 min at room temperature. The reaction was quenched with 125 mM glycine. Fixed cells were washed with cold PBS, scraped, centrifuged, resuspended in cold lysis buffer (15 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.5% N‑lauroylsarcosine, 0.1% sodium deoxycholate, 0.1% SDS), incubated for 10 min on ice and sonicated with a Bioruptor Pico for four cycles of 30 s on and 30 s off. ChIP-seq experiments were performed as previously described in Koenecke et al 2017 "Drosophila poised enhancers are generated during tissue patterning with the help of repression" with 107 mouse ESCs per ChIP. For each ChIP, 5 µg of the following antibodies were used: ɑ-Oct3/4 (Santa Cruz, sc-8628), ɑ-Sox2 (Santa Cruz, sc-17320), or ɑ-Nanog (Santa Cruz, sc-30328). At least two biological replicates were performed for each factor. Single-end sequencing was performed on either an Illumina HiSeq instrument (50 cycles) or NextSeq 500 instrument (75 cycles) according to manufacturer’s instructions. Sequencing libraries were prepared for sequencing using standard Illumina protocols The ChIP-nexus procedure and data processing were performed as previously described in Johnston et al 2015 except that the ChIP-nexus adaptor mix contained four fixed barcodes (ACTG, CTGA, GACT, TGAC). For each ChIP, 5 µg antibody was coupled to 50 µl of Dynabeads Protein A or Protein G (Invitrogen). The following antibodies were used: ɑ-Oct3/4 (Santa Cruz, sc-8628), ɑ-Sox2 (Santa Cruz, sc-17320), ɑ-Nanog (Santa Cruz, sc-30328), ɑ-Klf4 (R&D Systems, AF3158), ɑ-Klf4 (Abcam, ab106629), ɑ-Esrrb (Abcam, ab19331), ɑ-Pbx 1/2/3 (Santa Cruz, sc-888), and ɑ-Zic3 (Abcam, ab222124).
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NextSeq 500 |
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Data processing |
ChIP-nexus data processing steps: Random barcodes and fixed barcodes were trimmed off the reads and reassigned to FASTQ labels using nimnexus (v0.1.1). The adapters were then trimmed using cutadapt (v1.8.1). Reads were aligned with BWA (v0.7.13) using the command bwa aln -q 5 -l 32 -k to the mouse genome assembly mm10. Mapping stats were computed using SAMtools flagstat (v1.2). Mutant samples were aligned to a modified mm10 genome that accommodated the CRISPR changes. Reads were filtered using SAMtools view to remove unmapped reads and mates, non-primary alignments, reads failing platform or vendor quality checks, and PCR or optical duplicates (-F 1804). Low quality reads (MAPQ < 30) were also removed. Reads aligned to the same position with the same barcode, CIGAR string and the SAM flag were de-duplicated using nimnexus dedup (v0.1.1). The final filtered BAM file was converted to tagAlign format (BED 3+3) using bedtools `bamtobed` (v2.26) (126). Cross-correlation scores were obtained for each file using phantompeakqualtools (v1.2). BigWig tracks containing the strand-specific number of aligned 5' read ends (pooled across all replicates) were generated using bedtools genomecov -5 -bg -strand <+/->, followed by bedGraph to BigWig conversion using UCSC bedGraphToBigWig. Peaks were called using MACS2 (v2.1.1.20160309) by shifting and extending the reads to obtain coverage tracks similar to ChIP-seq (shift=-75, extsize=150). Peaks overlaping the blacklisted regions listed in http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/mm10-mouse/mm10.blacklist.bed.gz were excluded. Peak calling was performed on different combinations of aligned reads from different replicate experiments as described in Landt et al. 2012 and as used in the ENCODE ChIP-seq pipeline. Finally, 1kb regions centered at peak summits from the "optimal set" of reproducible, ranked consistent peaks passing the Irreproducible Discovery Rate (IDR) threshold of 0.05 were used as the peak regions for the corresponding TF (i.e. peaks that are reproducible and rank consistently across replicates). ChIP-seq datasets were processed using the ENCODE ChIP-seq pipeline https://github.com/ENCODE-DCC/chip-seq-pipeline2/releases/tag/v1.2.2. The ChIP-seq pipeline is identical to the ChIP-nexus pipeline described above except that it uses the SPP peak caller and doesn't use barcodes for read de-duplication. Genome_build: mm10 Supplementary_files_format_and_content: BigWig tracks containing the strand-specific number of aligned 5' read ends (pos=+ strand, neg=- strand) were generated using bedtools genomecov -5 -bg -strand <+/->, followed by bedGraph to BigWig conversion using UCSC bedGraphToBigWig. Supplementary_files_format_and_content: Bed files contain the "optimal'' peaks passing the IDR threshold of 0.05 as described above.
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Submission date |
Sep 20, 2019 |
Last update date |
Nov 21, 2020 |
Contact name |
Julia Zeitlinger |
E-mail(s) |
jbz@stowers.org
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Organization name |
Stowers Institute for Medical Research
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Lab |
Zeitlinger Lab
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Street address |
1000 E 50th Street
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City |
Kansas City |
State/province |
MO |
ZIP/Postal code |
64110 |
Country |
USA |
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Platform ID |
GPL19057 |
Series (1) |
GSE137193 |
ChIP-nexus data for Oct4, Sox2, Nanog and Klf4 in mouse embryonic stem cells |
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Relations |
BioSample |
SAMN12797746 |
SRA |
SRX6879006 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4087822_mesc_Esrrb_nexus.idr-optimal-set.narrowPeak.gz |
323.2 Kb |
(ftp)(http) |
NARROWPEAK |
GSM4087822_mesc_Esrrb_nexus.idr-optimal-set.summit.bed.gz |
118.9 Kb |
(ftp)(http) |
BED |
GSM4087822_mesc_Esrrb_nexus_pooled.neg.bw |
277.2 Mb |
(ftp)(http) |
BW |
GSM4087822_mesc_Esrrb_nexus_pooled.pos.bw |
277.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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