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| Status |
Public on Oct 03, 2023 |
| Title |
cR_H9_EOS_d0_rep1, H3K27me3 |
| Sample type |
SRA |
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| Source name |
chemically reset cR-H9-EOS
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| Organism |
Homo sapiens |
| Characteristics |
cell line: chemically reset cR-H9-EOS
cell type: chemically reset hPSC
biological replicate: 1
day of_formative_transition: 0
culture condition: N2B27 and 2uM XAV939
histone modification: H3K27me3
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
500.000 to 1.000.000 cells were used for ChIP per sample. Cross-linking was done with 1% formaldehyde in DMEM added directly to cells in culture dishes for 8 min at room temperature. Quenching was performed using 0.1M glycine (final concentration). The cells were washed with ice-cold PBS and scrapped with PBS with protease inhibitor cocktail (Cat. 11697498001, Roche). Lysis was performed in LB1 buffer (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% Igepal CA-630, 0.25% Triton X100, protease inhibitors) by rotating for 10 min at +4C, followed by centrifugation at 2000g for 5 min. The pellet was incubated in LB2 buffer (10mM Tris-HCl pH8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, protease inhibitors) by rotating for 5 min at +4C, followed by centrifugation at 2000g for 5 min. The pellet was resuspended in a buffer containing 50mM Tris-HCl pH8.0, 10mM EDTA, 1% SDS, the sonication was performed for 45 cycles, 30-sec intervals on/off. Debris was removed by centrifugation, and 10% from the sonicated material was saved as input. Prior to immunoprecipitation, 5ug antibody and 100ul Protein A beads were pre-incubated overnight at +4C in a total volume of 250ul, adjusted with PBS with 5mg/ml BSA, followed by three washes using the same solution. Immunoprecipitation was done by combining the sonicated material with the bead-antibody complexes, in a total volume of 400ul adjusted with ChIP dilution buffer (10mM Tris-HCl pH8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, 1% Triton X100, protease inhibitors), by rotating overnight at +4C. After the incubation, the beads were washed 6 times with RIPA buffer (50mM HEPES-KOH pH7.5, 500mM LiCl, 1mM EDTA, 1% Igepal CA-630, 0.7% sodium deoxycholate) followed by one wash in TE (10mM Tris-HCl pH8.0, 1mM EDTA). Reverse-crosslinking was done in elution buffer containing 1% SDS, 100mM sodium bicarbonate, and 200mM NaCl at 65C for 9-15 hours. The input sample was also reverse-crosslinked. After this step, the beads were removed from the mixture, and the remaining DNA in the solution was treated with 8 ug RNAse A for 1h at 37C, followed by 80ug proteinase K for 2 hours at 55C.
DNA was purified using MinElute columns and then used for ChIP-Seq library preparation using the NEXTflex Rapid DNA-Seq Kit (NOVA-5144-02) according to the manufacturer's protocol with some minor modifications. Briefly, we amplified the final libraries with 4 cycles of PCR, then performed an AMPure XP-based size selection (0.5x to eliminate larger fragments followed by 1.8x to extract the smaller fragments), and then continued with 8 more PCR cycles on the size selected material. The final libraries were then cleaned up with 0.8x vol AMPure XP beads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
cR_H9_EOS_d0_R1.mLb.clN_H3K27me3.bigWig
cR_H9_EOS_d0_R2.mLb.clN_H3K27me3.bigWig
cR_H9_EOS_d0_R1_peaks_H3K27me3.xls
cR_H9_EOS_d0_R2_peaks_H3K27me3.xls
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| Data processing |
nf-core/chipseq v1.1.0 pipeline used to process the data
FastQC v0.11.8 used for quality control
TrimGalore v0.5.0 used for trimming low quality bases + adapter sequences
BWA BWA v0.7.17-r1188 used for read mapping
Samtools v1.9 used for sorting bam files
BEDTools v2.27.1 used for converting sorted bam files into bedgraph files
MACS2 v2.1.2 used for calling peaks
Assembly: hg38
Supplementary files format and content: bigWig, txt files with MACS2 peaks, included in tar archives on the series record
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| Submission date |
Nov 21, 2022 |
| Last update date |
Oct 03, 2023 |
| Contact name |
Laura Biggins |
| E-mail(s) |
laura.biggins@babraham.ac.uk
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| Organization name |
The Babraham Institute
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| Department |
Bioinformatics
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| Street address |
Babraham Research Campus
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| City |
Cambridge |
| ZIP/Postal code |
CB22 3AT |
| Country |
United Kingdom |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE218510 |
Epigenetic dynamics during capacitation of naïve human pluripotent stem cells [ChIP-seq] |
| GSE218512 |
Epigenetic dynamics during capacitation of naïve human pluripotent stem cells |
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| Relations |
| BioSample |
SAMN31825341 |
| SRA |
SRX18337878 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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