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| Status |
Public on Mar 28, 2017 |
| Title |
XL9 IgG-Rb rep B |
| Sample type |
SRA |
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| Source name |
neural progenitor cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: ES-derived neural progenitor cells
cell line: WT26
strain: C57BL/6x129sV
clip antibody: IgG-Rb (Santa Cruz, sc-2027m lot H2814)
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| Treatment protocol |
The feeder-independent mouse male ES cell line WT26 (a kind gift from the lab of T. Jenuwein), was grown on gelatin-coated dishes in ES cell culture media KnockOut-DMEM supplemented with 1% L-glutamine, 1% penicillin/streptavidin, 1% Non- essential amino acids, 1% Sodium Pyruvate, 1% 2-mercaptoethanol. All ESC media contained 15% FBS and 1000 U/ml (for feeder free) or 2000U/ml (for feeder-dependent) of leukemia inhibitory factor (LIF). NPC lines were derived from previously mentioned ES cell lines and cultured in N2B27 medium (StemCell Sciences) containing 10 ng/mL EGF and FGF (Preprotech) on gelatin-coated flasks. The cells were kept in a humidified incubator at 37°C and 5% CO2 and were passed when reaching 80-90% confluency.
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| Growth protocol |
Mouse ES cells were differentiated into neuronal progenitor cells. In brief, ESC (1x106 cells) were deprived of feeders and plated on 0.1 % gelatin-coated dishes in N2B27 medium and grown for 7 days. During that period of time, the medium was changed every day. Next, cells were dissociated from the plate using accutase and approximately 3 x 10e6 cells were plated in a petri dish to induce formation of embryoid bodies (in N2B27 medium supplemented with 10 ng/ml EGF and FGF2). After 72 hours, embryoid bodies were transferred to 0.1 % gelatin-coated dishes and allowed to attach to the bottom leading to expansion of NPC cells. NPC lines were maintained in N2B27 medium supplemented with EGF and FGF2 (10ng/ml each), on 0.1 % gelatin- coated flasks.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells are crosslinked with 0.15mJ/cm2 UV-C irradiation, lysed with 1xNLB and homogenized by waterbath-sonication. Target protein of interest is pulled-down with paramagnetic beads pre-coupled to antibodies against the protein of interest.
After a brief RNAseI-digestion, RNA 3'-ends are healed with T4 PNK. Custom-made, barcoded adapters are ligated using T4 RNA Ligase 1 or T4 RNA Ligase 2KQ (if pre-adenylated adapters are used) for 1 hr at 25˚C. Custom FLASH adapters contained two barcodes and random nucleotides adjacent to the 3'-adapters according to the pattern NNBBNTTTTTTNN (N: random tag nucleotide, T: tag nucleotide, B: RY-space tag nucleotide). Random tags are used to merge PCR-duplicates, regular tags are used to specify the pulldown condition, and semi-random RY-space tags are used to distinguish the biological replicates (RR: replicate A, YY: replicate B, R: purine, Y: pyrimidine). Excess adapters are washed away, negative controls (IgG) are mixed with experimental controls and RNA is isolated with Proteinase K treatment and column purification. Isolated RNA is reverse-transcribed and RNaseH-treated. cDNA is column-purified and circularized with CircLigase for 2-16hrs. Circularized cDNA is directly PCR amplified, quantified with Qubit / Bioanalyzer and sequenced on Illumina NextSeq 500 in paired-end mode.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
3'-tag: TTAGCC, 3'-RY-space tag: YY
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| Data processing |
Adapters were trimmed using Flexbar (v2.5).
Libraries were demultiplexed using bctools (https://github.com/dmaticzka/bctools, v0.2.0) and Flexbar (v2.5).
Possible readthroughs into the barcoded regions were removed by clipping 13 nt from the 3'-ends of first mate reads.
Reads were mapped to reference genome hg19 using bowtie2 (v2.2.6) with parameters --very-sensitive --end-to-end --no-mixed --no-discordant --maxins 500.
Alignments of uniquely mapped reads were extracted and used to determine crosslinking events as previously described (Ilik, I. A. et al. Tandem Stem-Loops in roX RNAs Act Together to Mediate X Chromosome Dosage Compensation in Drosophila. Mol. Cell 51, 156–173 (2013)) with bctools (https://github.com/dmaticzka/bctools, v0.2.0).
Peaks for DHX9-Rb were called with PEAKachu (https://github.com/tbischler/PEAKachu/, v0.0.1alpha2) using parameters --pairwise_replicates, --max_insert_size 100, -m 0, -n manual, and --size_factors 1 1 0.75 0.75, with the IgG control as experimental background.
Genome_build: mm10
Supplementary_files_format_and_content: bed files containing coordinates of crosslinking event alignments with id of a representative read in name column and number of merged PCR-duplicates in score column. bigWig profiles of crosslinking event alignments for each strand. PEAKachu peaks in gff format.
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| Submission date |
Nov 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Asifa Akhtar |
| E-mail(s) |
akhtarlab_data@ie-freiburg.mpg.de
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| Organization name |
Max Planck Institute of Immunobiology and Epigenetics
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| Department |
Chromatin Regulation
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| Lab |
Akhtar Lab
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| Street address |
Stuebeweg 51
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| City |
Freiburg |
| ZIP/Postal code |
79108 |
| Country |
Germany |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE85164 |
DHX9 suppresses spurious RNA processing defects originating from the Alu invasion of the human genome |
| GSE89598 |
DHX9 suppresses spurious RNA processing defects originating from the Alu invasion of the human genome [XL9 DHX9 FLASH CLIP-seq] |
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| Relations |
| BioSample |
SAMN05990896 |
| SRA |
SRX2332603 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2385223_XL9_IgG_Rb_repB.bed.gz |
127.5 Kb |
(ftp)(http) |
BED |
| GSM2385223_XL9_IgG_Rb_repB_minus.bigWig |
66.8 Kb |
(ftp)(http) |
BIGWIG |
| GSM2385223_XL9_IgG_Rb_repB_plus.bigWig |
69.5 Kb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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