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Sample GSM5570324

Query DataSets for GSM5570324

Status

Public on Aug 10, 2023

Title

9sCa: ESCs_MSL2KO1_RNAseq_rep3

Sample type

SRA

Source name

Embryonic stem cells

Organism

Mus musculus

Characteristics

strain: 129S1(MAT)*CAST(PAT)
gender: Female
genotype: MSL2KO1
library type: RNAseq
replicate: rep3

Treatment protocol

For the MSL2 knockout generation, two guide RNAs (gRNAs) were cloned into a PX-459 derivative (pSpCas9(BB)-2A-Puro) plasmid (Addgene 62988), and a split puromycin resistance or GFP-positive approach for selection was used (Flemr and Bühler 2015). Male and female ES cells and NPCs were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. After 48 hours, the cells were sorted based on their GFP expression status using a MoFlo XDP Cell Sorter (Beckman Coulter). Genomic DNA from single clones was isolated by resuspending cells in lysis buffer (10 mM Tris pH 8.0, 0.5 mM Ethylenediaminetetraacetic acid (EDTA), 0.5% Triton X-100) supplemented with Proteinase K (Thermo Scientific), and incubated at 55°C for 1 hour. Genomic DNA was collected after centrifugation at 16,000 g for 10 minutes. Polymerase chain reaction (PCR) was carried out using the 2X PCR Master Mix (Qiagen) at a final volume of 10μL. Successful knockout was verified using CloneJet PCR Cloning (Thermo Scientific) and BigDye Direct Sanger Sequencing (Thermo Scientific) kits according to the manufacturer’s instructions.

Growth protocol

Male mouse ES cells, obtained from Anne Ferguson-Smith (University of Cambridge), were cultured in NDiff 227 medium (Takara) supplemented with LIF (Millipore), 2i inhibitors (PD0325901 and CHIR99021, Stemgent). Female mouse ES cells, obtained from Edith Heard (EMBL Heidelberg), were cultured in KnockOut-DMEM (KO-DMEM, Gibco) supplemented with 15% KnockOut Serum Replacement (KOSR, Gibco), GlutaMAX (Gibco), non-essential amino acids (Gibco), sodium pyruvate (Gibco), 2-Mercaptoethanol (Gibco), LIF, and 2i inhibitors. Male and female ES cells were differentiated into NPCs as previously described (Conti et al. 2005; Splinter et al. 2011). In brief, 1 million male or female ES cells were plated on 0.1% gelatin-coated plates in N2B27 medium, consisting of 1:1 DMEM/F-12 (Gibco) and Neurobasal medium (Gibco), supplemented with B27 (Gibco), GlutaMAX, 2-Mercaptoethanol, Apo-transferrin (Sigma), bovine serum albumin (Sigma), insulin (Sigma), putrescine (Sigma), progesterone (Sigma), and sodium selenite (Sigma). After 7 days, cells were dissociated and 3 million cells were plated on non-adherent culture dishes in the presence of 10ng/ml epidermal growth factor (EGF, Peprotech) and 10ng/ml basic fibroblast growth factor (FGF, Peprotech) to induce formation of embryoid bodies. After 3 days, the embryoid bodies were transferred to gelatin-coated plates to allow the expansion of NPCs.

Extracted molecule

total RNA

Extraction protocol

RNAseq: Total RNA was extracted using the Direct-zol RNA Miniprep Plus Kit (Zymo Research) according to the manufacturers’ instructions. Maxima First Strand cDNA Synthesis Kit (Thermo Scientific) was used to synthesize cDNA from total RNA according to the manufacturers’ instructions. Quantitative reverse transcription PCR was carried out on Roche LightCycler II using the Faststart SYBR Green Master (Rox) mix (Roche) at a final volume of 10μL.
TT-seq: In brief, 10 million NPCs were incubated with N2B27 medium supplemented with 500 uM 4- thiouridine (4sU, Sigma) for 5 minutes at 37°C. Total RNA was isolated, fragmented using Bioruptor Plus for 1 minute (30s ON, 30s OFF, high setting), and incubated with Biotin-HPDP (Thermo Scientific) for 2 hours. MyOne C1 Streptavidin magnetic beads (Thermo Scientific) were used to immunoprecipitate labelled RNA before elution in 5% beta-mercaptoethanol (Roth). FInal RNA was cleaned up using the Oligo Clean and Concentrator Kit (Zymo Research) according to the manufacturer’s instructions. Libraries were prepared from 100 ng of high quality RNA using the Ovation Universal RNA-Seq System (Nugen) according to the manufacturer’s instructions.
ChIPseq: MSL2 WT and KO NPCs were covered with 1% formaldehyde in culture media on the plate and incubated at room temperature (RT) on a rocking plate for 10 min. Media was removed and cells were washed twice with 1xPBS supplemented with 1x protease inhibitor cocktail (Roche). Cells were scraped off the plate, collected in ice-cold 1xPBS and centrifuged at 500g for 5 minutes. After an additional 1xPBS wash, cell pellets were stored at -80°C until usage. Nuclei were isolated using the NEXSON based nuclei isolation protocol. Cell pellets were thawed on ice and resuspended in ice cold 1ml of lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% Igepal, 1x protease inhibitor cocktail). The cell suspension was then transferred into 1ml milliTUBE (Covaris) and sonicated in Covaris instrument (E220) for 30 sec at peak power 75W, duty factor 2% and 200 cycles/burst. Nuclei were pelleted at 1000g at 20°C for 5min. The cytoplasmic fraction in the supernatant was discarded and nuclear pellets were carefully resuspended in 0.5% SDS and incubated at room temperature for 10min. Sample was diluted 1:5 in the digestion buffer (Final concentration: 1.1% Triton, 1x CutSmart buffer, nuclease free H2O, 1x protease inhibitor cocktail). Chromatin of up to 1 million nuclei were digested in 250ul digestion buffer with Cvik1 (5U/100.000 cells, New England Biolabs) at 20°C for 16hrs, shaking at 800rpm. After the chromatin digestion, the nuclei were pelleted for 5min at 1000g and washed in cold nuclei wash solution (10mM Tris-HCl pH 8, 0.25% Triton, 0.2mg/ml BSA) and stored on ice. To confirm the enzymatic chromatin shearing efficiency, 5% of the nuclei solution was incubated together with a decrosslinking solution consisting of 2ul proteinase K, 1ul RNaseA and 4ul 5M NaCl at 37°C for 30min followed by a 2hr incubation at 65°C. DNA was purified using MinElute PCR purification kit (Qiagen) and analyzed on a Agilent Tapestation system (G2964AA) using the Agilent D1000 Screentape reagents.
ATACseq: In brief, nuclei of 50,000 cells per replicate were isolated in 50 μl of cold ATAC resuspension buffer (0.1% NP-40, 0.1% Tween-20, and 0.01% digitonin (Promega)) by incubating on ice for 3 minutes. After lysis, 1 ml of cold ATAC resuspension buffer containing 0.1% Tween-20 (without NP-40 or digitonin) was added, and nuclei were then centrifuged at 500g for 10 minutes. Transposition was carried out using 50 μl of transposition mix (25 μl 2X TD buffer (Illumina), 2.5 μl transposase (Illumina), 16.5 μl 1X PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water) at 37°C for 30 minutes in a thermomixer with shaking at 1000 rpm. Transposed DNA was collected using Zymo DNA Clean and Concentrator (Zymo Research) according to the manufacturer's instructions, and added to the PCR mix containing 25 μl of NEBNext Master Mix (New England Biolabs) and 5 μl of Index adapters (Illumina). Libraries were amplified using the following PCR program: 72°C for 5 minutes, 98°C for 30 seconds, then 10 cycles of: 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute. Library quality was analyzed using the Fragment analyzer (Agilent Technologies) prior to sequencing.
BSseq: Bisulfite-Seq was performed using the NEBNext Enzymatic Methyl-seq Kit (E7120S) according to the manufacturer’s instructions. In brief, genomic DNA (gDNA) was extracted from wild-type and MSL2 knockout NPCs using the QIAamp DNA Mini kit (Qiagen) according to the manufacturer’s instructions.Subsequently, 50ng of high quality gDNA was sheared for 120 seconds using an E220 Covaris to obtain fragments between around 300 base pairs in length. Following end prep and EM-seq adapter ligation, DNA was oxidised and denatured using sodium hydroxide. Libraries were amplified for 6 PCR cycles, and sequenced with 2x100bp paired-end reads on an Illumina NovaSeq6000 sequencer.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

ESCs_DEseq_basic_DEresults.tsv
ESCs_DEseq_allelic_DEresults.tsv
ESCs_DEgenes_classification.txt

Data processing

RNAseq and TTseq: The data are processed via snakePipes mRNA-seq pipeline (developing branch) (Bhardwaj et al., 2019). SnakePipes removed adapters and low quality bases (< Q20) using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. The trimmed reads are aligned with STAR (v2.7.4) (Dobin et al., 2013) to an ‘N-masked’ genome, which is a version of the mouse reference genome mm10 where all the polymorphicsites (SNPs) for Mus musculus CAST/EiJ and Mus musculus C57BL/6 (or 129S1/SvImJ) are masked by ambiguity nucleobase ‘N’ (Keane et al., 2011). The mapped reads are then passed to SNPSplit (0.3.4) (Krueger and Andrews, 2016) to generate allele-specific BAM files by separating the alignment into two distinct alleles (CAST/EiJ and C57BL/6 or 129S1/SvImJ) based on SNPs information downloaded from Mouse Genome Database (Keane et al., 2011). The aligned reads of standard and allele specific level are counted separately on Gencode GTF (m9) via featureCounts (v2.0.0) (Yang et al., 2014).
ChIPseq: The data are processed via snakePipes DNA-mapping and ChIP-seq pipeline (developing branch) (Bhardwaj et al., 2019). Adapters and low quality bases (< Q20) are removed using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. For all samples, reads are then mapped to the ‘N-masked’’ genome with Bowtie2 (2.3.5) (Langmead and Salzberg, 2012). Reads mapped on blacklisted regions from Encode Consortium are discarded. Duplicated reads are also filtered with Picard MarkDuplicates (1.65) (https://broadinstitute.github.io/picard/). In the end, only properly paired mapped reads and reads with mapping quality over 3 are kept for further analysis. SNPSplit (0.3.4) (Krueger and Andrews, 2016) is then used to generate allele-specific BAM files and deepTools (v3.3.2) (Ramirez et al., 2014) is used to check data quality. In the ChIP-seq part, peak calling is performed with MACS2 (v2.2.6) (Zhang et al., 2008) with options [--qvalue 0.001]. It is done with input as control on total and allele specific ChIP-seq signals respectively. CSAW (v1.20.0) (Lun and Smyth, 2015) is additionally used to calculate log2FC on peak regions between MSL2 KO and WT or between allele2 and allele1. Bigwig files are created with deepTools bamCompare (v3.3.2) (Ramirez et al., 2014), using input normalization method and log2ratio and subtract option.
ATACseq: The data are processed via snakePipes DNA-mapping and ATAC-seq pipeline (developing branch) (Bhardwaj et al., 2019). In the DNA-mapping part, adapters and low quality bases (< Q20) are removed using TrimGalore (v0.6.5) with parameters '-q 20 --trim-n'. For all samples, reads are then mapped to the ‘N-masked’’ genome with Bowtie2 (v2.3.5) (Langmead and Salzberg, 2012). Reads mapped on blacklisted regions from Encode Consortium are discarded. Duplicated reads are also filtered with Picard MarkDuplicates (v1.65) (https://broadinstitute.github.io/picard/). In the end, only properly paired mapped reads and reads with mapping quality over 3 are kept for further analysis. SNPSplit (0.3.4) (Krueger and Andrews, 2016) is then used to generate allele-specific BAM files and deepTools (v3.3.2) (Ramirez et al., 2014) is used to check data quality. In the ATAC-seq part, the BAM files are filtered to include only properly paired reads with appropriate fragment sizes (<150 bases). To identify open/accessible regions, peak calling is done with MACS2 (v2.2.6) (Zhang et al., 2008) with options [--qvalue 0.001], on total and allele-specific ATAC-seq signal respectively. CSAW (v1.20.0) (Lun and Smyth, 2015) is additionally used to calculate log2FC on peak regions between MSL2 KO and WT or between allele2 and allele1. Bigwig files are created with deepTools bamCoverage (v3.3.2) (Ramirez et al., 2014), using size factor calculated by deepTools multiBamSummary (v3.3.2).
BSseq: Raw fastq files were aligned against the ‘N-masked’ genome and deduplicated using Bismark (v0.22.3) (Krueger and Andreas, 2011). Resulting Alignments were allele-separated using SNPsplit with flags '--paired' and '--bisulfite'. Relevant quality metrics (conversion rates, genome coverage and GC bias) were calculated using MethylDackel version 0.6.0 (https://github.com/dpryan79/MethylDackel) and DeepTools (v3.5.0). Allele-specific differentially CpG methylated sites between MSL2 KO and WT or between allele2 and allele1 were called with DSS version 2.34.0.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files: log2 ratio or subtract value over the input sample (merged replicates) . bed: peak calling from MACS2 (merged replicates). RData: differential peak calling result from CSAW and differential CpG methylation sites from DSS. tsv: differential expression analysis from DESeq2. txt: Differentially expression genes further classification into five cateogories.

Submission date

Sep 09, 2021

Last update date

Aug 10, 2023

Contact name

Asifa Akhtar

E-mail(s)

akhtarlab_data@ie-freiburg.mpg.de

Organization name

Max Planck Institute of Immunobiology and Epigenetics

Department

Chromatin Regulation