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Sample GSM4035069

Query DataSets for GSM4035069

Status

Public on Jun 23, 2020

Title

Dppa_DKO_serum_43_C

Sample type

SRA

Source name

Dppa2/4 DKO clone 43

Organism

Mus musculus

Characteristics

genotype: Dppa2/4 DKO
cell type: embryonic stem cell
culture conditions: serum/LIF
strain: CRISPR targetted E14 ESCs, contains MERVL::tdTomato reporter
chip antibody: N/A

Treatment protocol

Stable overexpression cell lines were generated using Lipofectamine 2000 on preplated cells and resistant cells selected with appropriate antibiotics for at least 1 week before sorting single cells into 96 well plates by flow cytometry using a BD Aria III or BD Influx high-speed cell sorter, and clonal expansion. CRISPR double knockout ESC line (clone 43) was described previously (M. Eckersley-Maslin et al., 2019), and additional CRISPR double knockout ESC lines (clone 37 and clone 53) generated as previously described (M. Eckersley-Maslin et al., 2019). For embryoid bodies, 2x106 mESCs were cultured on 10cm low-attachment dishes in standard ESC medium containing all described components except LIF.

Growth protocol

E14 mouse ESCs were grown using standard serum/LIF culture conditions (DMEM, 4,500 mg/L glucose, 4 mM L-glutamine, 110 mg/L sodium pyruvate, 15% fetal bovine serum, 1 U/mL penicillin, 1 mg/mL streptomycin, 0.1 mM nonessential amino acids, 50 mM b-mercaptoethanol, 103 U/mL LIF) at 37 degrees Celsius in normal oxygen, feeder-free on gelatin coated plates.

Extracted molecule

total RNA

Extraction 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.
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).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

DNaseI treated total RNA
Processed_data_ESC_RNAseq_RPM_allgenes.txt

Data processing

EB RNA-seq raw FastQ data were trimmed with Trim Galore (version 0.4.4, default parameters) and mapped to the mouse GRCm38 genome assembly using Hisat2 version 2.1.0. Stable overexpression clone data used Trim Galore v0.5.0_dev and HiSat2 v2.1.0.
ChIP-seq Raw FastQ data were trimmed with Trim Galore v0.6.1 and aligned to mouse GRCm38 genome using Bowtie 2 v 2.3.2.
ATAC-seq Raw FastQ data were trimmed with Trim Galore (v0.5.0_dev) using standard parameters and aligned using Bowtie 2 v2.3.2.
NOME-seq Raw fastQ data were trimmed using Trim Galore version 0.4.4 using standard parameters, reads aligned using Bismark v0.18.2
All downstream data processing was performed using SeqMonk software (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/).
Genome_build: mm10
Supplementary_files_format_and_content: The ChIP and ATAC is enrichment RPM over 1kb running windows. The RNA-seq is all genes RPM and the NOME-seq is 100CpG running window % methylation. The files were generated using SeqMonk (exporting an annotated probe report).

Submission date

Aug 14, 2019

Last update date

Jun 23, 2020

Contact name

Steven William Wingett

E-mail(s)

steven.wingett@mrc-lmb.cam.ac.uk

Organization name

MRC Laboratory of Molecular Biology

Department

Cell Biology