inducible shRNA cells treated with dox for 7 days then dox washout for 14 days
cell type
embryonic stem cell
culture conditions
serum/LIF
strain
E14 ESCs containing inducible shRNA hairpin against Dppa2
chip antibody
anti-H3K4me3 Abcam ab8580
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
RNAseq: Total RNA was isolated using TriReagent (Sigma) or RNA-DNA allprep columns (Qiagen) 0.5-1mg DNAse treated (Thermo Fisher EN0525). ChIPseq: Ten million cells were fixed in 1% formaldehyde (Fisher Scientific 28906) in DMEM (Invitrogen 41966-052) for 10 minutes, quenched in 0.125M glycine, and scraped off cell culture dishes using cell scrapers. Cells were lysed with buffers LB1 (50mM Hepes-KOH (pH 7.5), 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% Igepal CA-630, 0.25% Triton-X 100), LB2 (10mM Tris-HCl (pH 8.0), 200mM NaCl, 1mM EDTA, 0.5mM EGTA), and LB3 (10mM Tris-HCl (pH 8.0), 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1x Protease Inhibitors (Roche)) consecutively, before fragmenting chromatin by sonicating for 30 cycles of 30s ON high /30s OFF (Diagenode Bioruptor). For each ChIP, 5µg antibody (anti-Ash2l Bethyl Labs A300-489, anti-Ezh2 CST D2C9, anti-H2A.Z Abcam ab4174, anti-H3K4me3 Abcam ab8580, anti-H3K27me3 Active Motif AM39155, anti-IgG Abcam ab125938, anti-Ring1b CST D22F2) was pre-bound to protein G dynabeads (Invitrogen) and blocked with 0.5% BSA in PBS. Sonicated DNA was added to antibody-bound beads with 10% Triton-X 100 and incubated overnight at 4°C on a rotator. Beads and DNA were washed 6 times with RIPA buffer (50mM HEPES-KOH (pH 7.6), 1mM EDTA, 0.7% Na-Deoxycholate, 1% Igepal CA-630, 0.5M LiCl), followed by one wash in 1x TBS and overnight incubation at 65°C with elution buffer (50 mM Tris- HCl, pH 8; 10 mM EDTA; 1% SDS). For precipitation of chromatin, samples were treated with RNase A and Proteinase K. DNA was purified using MinElute PCR purification columns (Qiagen 28006) and eluted in 30µL of TE. DNA was quantified using HS DNA Qubit (ThermoFisher) or PicoGreen (ThermoFisher) assays. ATACseq: 10,000 cells were trypsinised and immediately processed for ATACseq. NOMEseq: Cells were fixed on 15cm plates wih Fixation solution (Active-Motif) for 10minutes at room temperature then processed according to Active Motif NOME-seq kit protocol. amplicon bisulfite: Genomic DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen) or the AllPrep DNA/RNA Micro Kit (Qiagen) according to manufacturer's instructions and eluted into TE buffer or H2O. RNAseq: Opposite strand-specific polyA RNA libraries were made using 1 µg of DNase- treated RNA at the Sanger Institute Illumina bespoke pipeline and sequenced using the Illumina HiSeq2 platform. ChIPseq: libraries generated using MicroPlex Library Preparation Kit (Diagenode) following the manufacturer's instructions. Libraries were quality controlled using bioanalyser HS DNA chips (Agilent) and single-end 50bp reads sequenced using the Illumina HiSeq2 sequencing platform. ATACseq: ATAC-seq libraries were performed as previously described (Buenrostro, Wu, Chang, & Greenleaf, 2015) with the following modification to use on 10,000 cells: initial transposition reaction was performed with 10,000 cells, 10ml 2x TD buffer, 0.5ml Tn5 enzyme and 9.5ml H2O for 30minutes at 37 degrees Celsius. A total of 15 cycles of amplification were used. Two technical replicates were performed per clone, with 3 WT and 3 Dppa2/4 DKO clones used (total of 12 samples). NOMEseq: Whole genome bisulfite libraries were generated using NOME-seq kit from Active Motif (103946) according to manufacturer's instructions with 6 cycles of amplification for 2 WT clones (clones 57 and 58) and 1 Dppa2/4 DKO clone (clone 43). amplicon bisulfite: 1μg of genomic DNA was bisulfite converted using the EZ DNA methylation Gold kit (Zymo Research) according to manufacturer's instructions with either magnetic bead or column clean-up and eluted/resuspended in elution buffer. Amplicons of interest were amplified using 2x KAPA HiFi Uracil+ ReadyMix using primers containing UMI for deduplication and PCR adapters for a second round of PCR to adapt sample-specific barcodes and adapters for Illumina Sequencing. Libraries were sequenced using the Illumina MiSeq platform with a 10% PhIX spike-in and paired-end 250bp reads. : RNA-seq or ChIP-seq or NOME-seq or amplicon bisulfite-seq