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Sample GSM5029099 Query DataSets for GSM5029099
Status Public on Jul 25, 2023
Title RL383_NeuN_invitro_methylC_rep1
Sample type SRA
 
Source name mESC-derived NeuN positive neurons day 38
Organism Mus musculus
Characteristics cell line: G4
cell type: mESC-derived NeuN positive neurons day 38
cell culture: in vitro
age: 38 days
Treatment protocol High potassium-mediated depolarisation was performed by washing cells with low K+ buffer, transferring to high K+ buffer for 5 min, washed again in low K+ buffer and either harvested directly for RNA, immediately fixed for ICC, or chased in growth medium for the appropriate times prior to fixation or RNA extraction.
Growth protocol mESC cells lines R1 and G4 were maintained on gamma-irradiated mouse embryonic fibroblasts in mESC medium with LIF. Cells were fed daily and split based on confluency. Neural differentiation was initiated by dissociating the mESC colonies using Accutase and excess feeder cells were removed from the cell suspensions by panning on gelatin-coated plates. Dissociated cells were counted and transferred to ultra-low attachment cell culture plates at a dilution of 220,000 cells/ml, in differentiation medium. Neural induction was continued for 8 days, medium was changed every two days, and supplemented with 5 µM retinoic acid for the final 4 days. Cell aggregates were dissociated using Accutase, single cell selected through a 40 µm cell strainer, and plated onto poly-ornithine / laminin-coated plates in N2 medium. After 2 days, cells were changed into N2B27 which was changed every 4 days for 12 days, following which 50% media changes were performed for the remaining culture time.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA: Quiagen Blood and Tissue DNA Extraction kit with extended Proteinase K and RNAsA treatment steps. Total RNA: hot trizol RNA extraction on NeuN-sorted nuclei and DNAse I treatment.
Lucigen Nxseq Ampfree Low DNA Library Kit and Zymo EZ DNA Methylation Direct Kit for MethylC libraries. TruSeq stranded mRNA library prep kit with TruSeq unique dual index adapters for RNA-seq libraries.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
Description ESC-derived wild type neural differentiation, sorted NeuN positive fraction
Data processing Library strategy: Methyl-C-Seq
MethylC adapter trimming using Methpipe pipeline as described in Lister et al Science 2013
mapping MethylC reads to mm10 genome using Bowtie as described in Lister et al Science 2013
merging mapped runs, convert to .slam, sort, collapse, trim reads using Methpipe pipeline as described in Lister et al Science 2013
stack creation and hammer correction using Methpipe pipeline as described in Lister et al Science 2013
make allC tables and count global statistics using Methpipe pipeline as described in Lister et al Science 2013
convert stacks to bedgraph files using simple awk script
generate bigwigs using bedgraphtobigwig v4
calling differentially methylated regions between in vitro and in vivo MethylC datasets using HOME v. 1.0
Genome_build: mm10
Supplementary_files_format_and_content: stacks.txt files containing all mapped methylated and total cytosines, bigWigs for coverage containing all mapped cytosines, bigWigs for mC containing percentage of methylated cytosines for contexts CG and CH
Supplementary_files_format_and_content: DMRs.txt files contain location of DMRs and confidence scores with positive values indicating higher methylation in vitro and negative values indicating higher methylation in vivo.
 
Submission date Jan 21, 2021
Last update date Jul 25, 2023
Contact name Ryan Lister
E-mail(s) ryanlister@gmail.com
Phone 61864884407
Organization name The University of Western Australia
Street address 35 Stirling Highway
City Perth
State/province WA
ZIP/Postal code 6009
Country Australia
 
Platform ID GPL24247
Series (1)
GSE137098 Embryonic stem cell-derived neurons as a model system for epigenome maturation during development
Relations
BioSample SAMN17478435
SRA SRX9910243

Supplementary file Size Download File type/resource
GSM5029099_RL383_NeuN_invitro_rep1.CG.coverage.bigWig 275.1 Mb (ftp)(http) BIGWIG
GSM5029099_RL383_NeuN_invitro_rep1.CH.coverage.bigWig 4.5 Gb (ftp)(http) BIGWIG
GSM5029099_RL383_NeuN_invitro_rep1.mCG.percent.bigWig 273.7 Mb (ftp)(http) BIGWIG
GSM5029099_RL383_NeuN_invitro_rep1.mCH.percent.bigWig 4.4 Gb (ftp)(http) BIGWIG
GSM5029099_RL383_NeuN_invitro_rep1_methylC.stacks.txt.gz 9.8 Gb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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