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Sample GSM5920433

Query DataSets for GSM5920433

Status

Public on Oct 31, 2022

Title

Experiment1 shEmpty

Sample type

SRA

Source name

mESC

Organism

Mus musculus

Characteristics

strain: C57BL/6
transduction: shEmpty
time-point: Samples collected 5 days post puromycin selection
full name: 1E9D5_S14

Treatment protocol

mESCs were transduced with shRNA expressing lentiviral vectors. Puromycin selection was performed 2 days post transduction overnight (and control cells verified to die) and samples harvested 5 days post selection. Cells tranduced with GFP shRNA expressing lentiviral vectors were sorted on day 2 post transduction and harvested 5 days later.

Growth protocol

ES3 mESCs were cultured in Glasgow Minimum Essential Medium (GMEM, Sigma) supplemented with 100 U/ml penicillin/streptomycin, 10% fetal calf serum (FCS, Life Technologies), 2mM L-glutamine (Gibco, Thermo Fisher Scientific), 1mM Sodium pyruvate (Sigma), 1x MEM non-essential amino acids (Gibco, Thermo Fisher), 0.1mM 2-Mercaptoethanol (Life Technologies) and 1,000 units/ml Leukaemia inhibitory factor (LIF, Chemicon) and grown at 5% CO2 at 37 °C. mESC lines were split 1:4 every 2 days using accutase or trypsin.

Extracted molecule

total RNA

Extraction protocol

RNA-seq - Total RNA was extracted using RNeasy Micro kit columns (Qiagen) and DNase treated according to the manufacturer’s instructions (Ambion AM1907). RNA was quality checked on a 4200 Tapestation using the RNA ScreenTape assay (Agilent Technologies, Wokingham, UK) and the RNA concentration measured using a Qubit RNA Broad Range kit (Life Technologies, Paisley, UK)
CUT&RUN - We followed the EpiCypher® CUTANATM CUT&RUN Protocol (v1.6) (https://www.epicypher.com/resources/protocols/cutana-cut-and-run-protocol/). Briefly, CUT&RUN was performed using 100,000 sorted GFPBright cells (per antibody/sample combination). Cells were washed twice [20mM HEPES pH 7.5, 150mM NaCl, 0.5mM spermidine, 1× protease inhibitors (cOmplete™, Mini, EDTA-free, Roche)], attached to Concanavalin A beads (BioMag®Plus, Generon) which were pre-activated in activation buffer (20mM HEPES pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2). Sample and bead slurry was resuspended in antibody buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM Spermidine, 1× protease inhibitors, 0.01% w/v digitonin, 2mM EDTA) containing primary antibody. 1ug of anti-H3K9me3 (Abcam, ab8898) or anti-H3K27ac (Abcam, ab4729) was incubated overnight at 4°C with gentle shaking. IgG control Ab (Millipore, 12-370) alone was used in parallel. The samples were washed 3x in cold digitonin buffer (20 mM HEPES pH 7.5, 150mM NaCl, 0.5mM Spermidine, 1× Roche complete protease inhibitors, 0.01% digitonin). pA-MNase (2.5μl per tube, CUTANA™ pAG-MNase, Epicypher) was added incubated with the bead-cell slurry at room temperature for 10min. The beads were washed twice and resuspended in cold digitonin buffer. The pA-MNase was activated by addition of 2mM CaCl2 (final) and incubating at 4°C for 2h. The reaction was quenched using stop buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 50μg/ml glycogen, 50μg/ml RNase A), vortexing and incubating for 10 min at 37°C. A magnetic rack was used to separate cells and beads from the supernatant which was purified with a MinElute PCR Purification Kit (Qiagen).
ChIP - ES3 mESCs were transduced with ZFP819 and ZFP809-expressing lentiviral vectors (pRRLSIN.cPPT.PGK-ZFP.WPRE vectors with a triple HA tag). Cells were washed twice (in PBS + 2% FCS), counted to normalize by cell number, cross-linked (10 min rotation in 1% formaldehyde), quenched with glycine (at 125 mM on ice), washed three times (PBS), and snap-frozen at 107 cells per tube. Cells were resuspended in sequential ChIP lysis buffers and then in 900ul of sonication buffer on ice (10 mM Tris at pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% NaDOC, 0.25% NLS, and protease inhibitors). Where a Covaris sonicator was used, 900ul was sonicated in one covaris glass tube with the settings: 20% duty cycle, intensity 5, 200 cycles/burst, 30 min. For sonication with a Bioruptor Pico, 300ul aliquots per 1.5ml tube were sonicated1. Immunoprecipitations were performed in duplicates as described2 using an HA Ab (Biolegend, 901501) and DNA extracted (Qiagen MinElute PCR purification kit, Cat. 28004) for PCR and sequencing. See Supplementary Information for primers. 1. Tie, C.H. et al. KAP1 regulates endogenous retroviruses in adult human cells and contributes to innate immune control. EMBO Rep 19(2018). 2. Robbez-Masson, L. et al. The HUSH complex cooperates with TRIM28 to repress young retrotransposons and new genes. Genome Res (2018).
RNA-seq - 500 ng per sample was processed using KAPA’s stranded RNA HyperPrep RiboErase kit and sequenced on a NextSeq 500 instrument (Illumina Cambridge, Chesterford, UK) after pooling libraries in equimolar quantities, using a 2x 151 bp paired-end run, resulting in over 15 million reads per sample.
CUT&RUN - Illumina sequencing libraries were prepared using a NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® and NEBNext® Multiplex Oligos for Illumina® (New England BioLabs). Enrichment of DNA fragments was achieved by PCR (12 cycles). Libraries pooled in equimolar quantities were sequenced on Novaseq6000 for 150 paired-end reads.
ChIP-seq - Libraries were prepared following the ChIP-Seq library preparation protocol (Illumina, IP-102-1001). Ligation products were purified on 2% E Gels Size Select (Invitrogen; fragments of 200 bp recovered), followed by enrichment of DNA fragments by PCR (18 cycles). Sequencing was performed with Illumina HiSeq 2500 in 100-bp single-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

mESCs transduced with empty pLKO.1 (Addgene #10878) lentiviral vector (known as shControl) (see Fernandes et al., 2022 for shRNA sequences)
Sup_Table_TEcounts.xlsx

Data processing

RNA-Seq and CUT&RUN fastq files were trimmed for quality using TrimGalore v0.4.1
For RNA-Seq samples, alignment of trimmed reads to mm10 version of the mouse genome was performed with Tophat 2.1.0 using the --max-multihits 100 option. The number of read counts per gene and repeat family was calculated using the multi option from Tecount. Differential expression analysis was performed with DESeq2. For further details see Fernandes et al., 2022.
For CUT&RUN samples, trimmed reads were aligned with STAR with the following parameters: --outFilterMultimapNmax 5000 --outSAMmultNmax 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.06. Genomic coverage in form of bigwig files was calculated using the genomecov tool from bedtools using 10000000 / number_of_mapped_reads as a scaling factor.
For ChIP-Seq samples, reads were mapped to the mm10 version of the mouse genome using Bowtie2. ChIP-seq enriched peaks were called by MACS2 (v.2.1.2), and the common peaks were merged using bedtools
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig files for genomic coverage normalised to number of mapped reads. Excel spreadsheet with results of differential expression analysis. Bed files with ChIP-Seq peaks

Submission date

Feb 27, 2022

Last update date

Oct 31, 2022

Contact name

Helen M. Rowe

E-mail(s)

h.rowe@qmul.ac.uk

Organization name

Queen Mary University of London

Department

Blizard Institute, Barts and The London School of Medicine and Dentistry