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| Status |
Public on Oct 01, 2022 |
| Title |
ES_ChIL1000C_IgG_1 |
| Sample type |
SRA |
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| Source name |
mES-cells
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| Organism |
Mus musculus |
| Characteristics |
protocol: ChIL-seq
assay: IgG
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIL-seq was performed following a modified protocol of the original ChIL-seq (PMID: 30532068). For ES cells, 100 or 1000 cells were plated on gelatin coated 96-well plates and let to attach for 16h in culture. Cells were then fixed with 1% formaldehyde in DMEM for 5 min and permeabilized with 1% Triton X-100 in PBS for 20min. Embryos were fixed using the same procedure. Samples were incubated in 2% BSA in PBS with anti-Pol II Ser5P (Abcam, ab5131) or anti-Pol II Ser2P (Abcam, ab5095) at 2μg/ml for 6h at room temperature, washed three times in 0.5% BSA in PBS and then incubated at 4 °C overnight in 2% BSA in PBS containing 500mM NaCl and the ChIL probe at 2μg/ml. After washing in PBS for ES-cells or PBS containing 0.1% BSA for embryos, samples were incubated in 50 µl Tn5 solution (50mM HEPES-KOH, pH 7.2, 0.1M NaCl, 0.1mM EDTA, 1mM dithiothreitol, 0.1% Triton X-100 and 10% glycerol) containing 0.5µl HActive™ Tn5 transposase (Glow Biologics, GBRP-TN5) for 10min at room temperature. Tn5-MEDS-B oligo (2 µM) was then added at 0.5 µl per well in 1× dialysis buffer (50mM HEPES-KOH, pH 7.2, 0.1M NaCl, 0.1mM EDTA, 1mM dithiothreitol, 0.1% Triton X-100 and 10% glycerol) and incubated for 60min at room temperature. Samples were then washed in 1× dialysis buffer and three times in PBS for ES-cells or PBS containing 0.1% BSA for embryos, incubated in 100 µl 1× TAPS-DMF buffer (10mM TAPS-NaOH, pH 8.5, 5mM MgCl2 and 10% N,N-dimethylformamide) for 1h at 37 °C, and washed at three times in PBS for ES-cells or PBS containing 0.1% BSA for embryos. To stop the Tn5 reaction, cells were incubated in 100 µl 0.2% SDS for 10min at room temperature or embryos were incubated in 0.1% BSA/PBS containing 50mM EDTA at 50 °C for 30 min. After washing at three times in PBS for ES-cells or PBS containing 0.1% BSA for embryos, samples were incubated in 50 µl reaction mixture (200U T4 DNA ligase, NEB, and 1.5U T4 DNA polymerase, NEB, in 1× T4 DNA ligase reaction buffer containing 0.1 mM dNTP) for 30min at room temperature. After discarding the solution, for ES cells, 100µl 0.2% SDS were added to remove DNA-bound enzymes, followed by incubation for 10min at room temperature and three washes in PBS. For embryos, 0.1% BSA/PBS containing 50mM EDTA was added for 30 min at 50 °C, and then washed at three times in 0.1% BSA/PBS. In situ transcription was then carried out using T7 RNA polymerase (800U per well; NEB) for 20h at 37 °C. The samples were treated with DNase I (2.5U per well; Thermo) for 30min at 37 °C and RNA was extracted using a Qiagen MinElute Cleanup kit.
Purified RNA was reverse-transcribed with SMARTScribe reverse transcriptase (Takara) following the manufacturer’s instructions and the resulting cDNA was amplified by Prime star GXL (Takara) by 14 cycles for 1000 cells with anti-Pol II Ser5P antibody, 15 cycles for 1000 cells with anti-Pol II Ser2P antibody, 16 cycles for 100 cells or embryos with anti-Ser5P antibody, or 17 cycles for 100 cells or embryos with anti-Ser2P antibody. After amplification, small fragments (<150bp) were removed using Gene read Size selection kit (Qiagen) and large size of fragments (>600bp) were removed using AMPure XP beads (Beckman Coulter). The amplified DNA was purified using AMPure XP beads and the quality of the libraries was assessed using a Bioanalyzer 2100 (Agilent Technologies). Samples were paired-end sequenced at PE150 on an Illumina NovaSeq 6000 platform
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: ChIL-seq
The mouse reference genome and annotations were downloaded from Ensembl (version GRCm38.dna.primary_assembly.fa and GRCm38.101.gtf).
Paired-end sequencing reads were trimmed using cutadapt (version 3.4) with parameters -a CTGTCTCTTATA -A CTGTCTCTTATA --pair-filter=any --minimum-length=20.
Trimmed reads were mapped to the reference genome using bowtie2 (version 2.3.5) with parameters --end-to-end --very-sensitive --no-unal --no-mixed --no-discordant -I 10 -X 500.
Aligned reads were filtered by quality using samtools (version 1.3) with parameter -q 12. Read pairs were read in to R (version 3.6.3) using readGAlignmentPairs from the GenomicAlignment package (version 1.22.0) and fragments were filtered for fragments that have both unique start and end positions.
Coverage vectors were created using the coverage function from GenomicRanges package (version 1.38.0) and were normalized by the number of fragments and multiplied by a million. Normalized coverage were exported as bigwig files using the rtracklayer package (version 1.46.0).
Genome_build: GRCm38
Supplementary_files_format_and_content: bigwig files of normalized coverage
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| Submission date |
Jan 31, 2022 |
| Last update date |
Oct 02, 2022 |
| Contact name |
Tamas Schauer |
| E-mail(s) |
tamas.schauer@helmholtz-munich.de
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| Organization name |
Helmholtz Zentrum München
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| Department |
Institute of Epigenetics and Stem Cells
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| Street address |
Feodor-Lynen-Straße 21
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| City |
Munich |
| ZIP/Postal code |
81377 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE195827 |
Characterization and function of transcriptional elongation during zygotic genome activation in mouse embryos [ChIL-seq] |
| GSE195840 |
Characterization and function of transcriptional elongation during zygotic genome activation in mouse embryos |
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| Relations |
| BioSample |
SAMN25521211 |
| SRA |
SRX13999649 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5851485_ES_ChIL1000C_IgG_1_norm_coverage.bw |
38.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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