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| Status |
Public on Dec 08, 2019 |
| Title |
GFP_PGCLC_noIAA_r2 |
| Sample type |
SRA |
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| Source name |
Primordial germ cell-like cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Primordial germ cell-like cells
treatment: No auxin
chip antibody: anti-GFP (abcam ab290)
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| Growth protocol |
PGC-competent 4i hESCs (WIS2-NANOS3-T2A-tdTomato) were cultured on irradiated mouse embryonic fibroblasts (MEFs) (GlobalStem) in 4i medium. Media were replaced every day. hESCs were passaged by single-cell dissociation using 0.25% Trypsin-EDTA (GIBCO). 10 μM ROCK inhibitor (Y-27632, TOCRIS) was added for 24 hours after passaging. To induce hPGCLCs, 4i hESCs were trypsinized, filtered and plated into 6-well EZSPHERE microplates (ReproCELL) (500,000 cells/well in 3 mL PGCLC medium). The plates were centrifuged at 300g for 3 minutes and placed into a 37 °C 5% CO2 incubator until embryoid body (EB) collection.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The following samples were included in the analysis (one replicate per condition): 4i hESCs (cl11 no IAA), 4i+IAA hESCs (cl11+IAA) and hPGCLCs (cl11 and cl21 pooled; no IAA). For each ChIP, 2.5 million unsorted 4i hESCs or 4 million unsorted hPGCLCs (D4 EBs containing 60-70% PGCLCs as assessed by flow cytometry) were used. The cells were washed in PBS, filtered through 50 μm strainer and fixed in 1% formaldehyde at RT for 10 min. Formaldehyde was quenched by adding 450 μl of 10x glycine at RT for 10 min, followed by centrifugation at 500 g, 4 ºC for 5 minutes. The cells were then washed twice in ice-cold PBS with protease inhibitor cocktail (PIC) and the pellets were snap frozen on dry ice and stored at -80 ºC. ChIP was performed using SimpleChIP Enzymatic Chromatin IP Kit (with Magnetic Beads) from Cell Signalling Technology (CST) following manufacturer’s recommendations with modifications.
ChIP-seq libraries were prepared using KAPA HyperPrep Kit following the manufacturer’s instructions. Briefly, the protocol contains the following steps: end-repair and A-tailing; sequencing adapter (index) ligation; product purification using AMPure beads; library amplification using KAPA real-time Library Amplification Kit (11 cycles were used for ChIP libraries and 7 for input libraries to achieve similar concentration range); product purification using AMPure beads. Libraries were then quantified by qPCR using NEBNExt Library Quant Kit for Illumina (NEB) on QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Fragment size distribution and the absence of adapter dimers were checked using Agilent TapeStation 2200 and High Sensitivity D1000 ScreenTape.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
The library sequence quality in demultiplexed fastq files was checked by FastQC (v0.11.5) and the low-quality reads and adaptor sequences were removed by Trim Galore (v0.4.1) using the default parameters.
The trimmed ChIP-seq reads were aligned to the human reference genome (UCSC hg38) by the Burrows-Wheeler Aligner (0.7.15-r1142-dirty).
Samtools (version: 1.3.1) was used to remove unmapped and low mapping quality reads (options: view -F 4 -q 20). Subsequently, duplicated reads and reads mapped to unlocalized contig (random), unplaced contigs (ChrUn) and blacklisted regions (obtained from http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/hg38-human/) were removed using Samtools rmdup.
Before peak calling, all libraries were downsampled to 25 million reads. Peaks were then called using macs2 callpeak against the corresponding inputs (options: -g 3e9 --keep-dup all --nomodel –extsize 157).
Genome_build: hg38 (GRCh38)
Supplementary_files_format_and_content: *_peaks.narrowPeak: Peak files are in the macs2 narrowpeak format (refers to https://github.com/taoliu/MACS).
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| Submission date |
Oct 09, 2019 |
| Last update date |
Dec 10, 2019 |
| Contact name |
Walfred Tang |
| E-mail(s) |
walfredtang@gmail.com
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| Organization name |
University of Cambridge
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| Department |
Gurdon Institute
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| Lab |
Azim Surani
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| Street address |
Tennis Court Road
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| City |
Cambridge |
| ZIP/Postal code |
CB2 1QN |
| Country |
United Kingdom |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE138674 |
A critical but divergent role of PRDM14 in human primordial germ cell fate revealed by inducible degrons [ChIP-seq] |
| GSE138675 |
A critical but divergent role of PRDM14 in human primordial germ cell fate revealed by inducible degrons |
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| Relations |
| BioSample |
SAMN13005859 |
| SRA |
SRX6973254 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4115966_GFP_PGCLC_noIAA_r2_peaks.narrowPeak.gz |
100.8 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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