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Sample GSM3290274 Query DataSets for GSM3290274
Status Public on Jul 18, 2018
Title reprogramming with siMyc (for cMyc, nMyc, lMyc), day 3
Sample type SRA
 
Source name Mbd3f/- OSK2nd cell line, day 3 of reprogramming
Organism Mus musculus
Characteristics cell line: Mbd3f/- + OSK 2nd cell line
stage of reprogramming: day 3
Treatment protocol For secondary Mbd3f/- OSK2nd production, primary MEFs from Mbd3flox/- chimeric mice were infected with FUW-TetO-STEMCCA-OKS-mCherry and FUW-M2rtTA. IPS cells were isolated and injected into BDF2 blastocysts for the isolation of secondary MEFs. Secondary MEFs were transfected at day -3 and again at day 0 (starting reprogramming by adding DOX) with siRNA for cMyc, lMyc, nMyc or control (Stealth siRNA- mix of 3 as indicated in the paper) with RNAiMAX (Invitrogen). For molecular analysis, cells were collected at day 3 and day 7 as indicated.
Growth protocol Maintenance and reprogramming (Day 3 and beyond) of murine naïve pluripotent cells were conducted in bovine serum-free N2B27-based media or KSR media: N2B27- based media: 500ml KO-DMEM (Invitrogen), 5ml N2 supplement (Invitrogen; 17502048), 5ml B27 supplement (Invitrogen; 17504044), 15% knockout serum replacement (Invitrogen – 10828), 1mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), penicillin-streptomycin (Invitrogen), 5mg/ml BSA (Sigma). KSR media: 500 ml DMEM (Invitrogen) , 1mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), penicillin- streptomycin (Invitrogen). Naïve conditions for murine PSCs included 10μg recombinant human LIF (Millipore; LIF1005). Where indicated 2i was added 48 hours after OSKM induction: small-molecule inhibitors CHIR99021 (CH, 3μM- Axon Medchem) and PD0325901 (PD 0.2 or 1μM - Axon Medchem).
Extracted molecule polyA RNA
Extraction protocol Total RNA was isolated from indicated cell lines, and extracted from Trizol pellets by Direct-zol RNA MiniPrep kit (Zymo) according to manufacturer’s manual.
RNA was extracted from Trizol pellets, and utilized for RNA-Seq by TruSeq RNA Sample Preparation Kit v2 (Illumina) according to manufacturer’s instruction.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description Myc_pert.fpkm.txt.gz
Data processing We used Illumina CASAVA 1.8.2 software to generate fastq files.
Tophat software version 2.0.10 was used to align reads to mouse mm10 reference genome
FPKM values were calculated over all genes in mm10 assembly GTF (UCSC, December 2011), using cufflinks version 2.2.1.
FC was calculated in compare to MEF siControl, replicate 1.
Genome_build: mm10
 
Submission date Jul 17, 2018
Last update date Jul 18, 2018
Contact name Noa Novershtern
E-mail(s) noa.novershtern@weizmann.ac.il
Organization name Weizmann Institute of Science
Department Molecular Genetics
Street address Weizmann Institute
City Rehovot
ZIP/Postal code 7610001
Country Israel
 
Platform ID GPL19057
Series (2)
GSE102348 High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems [RNA-Seq]
GSE102518 High-Resolution Dissection of Conducive Somatic Cell Reprogramming to Naïve Ground State Pluripotency in Mbd3/NuRD and Gatad2a/NuRD Depleted Systems
Relations
BioSample SAMN09684334
SRA SRX4401527

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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