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Sample GSM5348215 Query DataSets for GSM5348215
Status Public on Nov 24, 2021
Title RNA-betacateninKD2_lowdensity_r1
Sample type SRA
 
Source name Mouse embryonic stem cells, differentiated
Organism Mus musculus
Characteristics cell type: J1 mouse embryonic stem cells
treatment: shRNA against Ctnnb1
Treatment protocol Dox was used at the indicated concentrations for last 48 hr (included in the 72 hr of differentiation). Inducible expression was achieved through transfection with pSB vector containing FLAG-BIO-tagged Esrrb, Tcfl7l1, or beta-catenin in BirA-expressing cells, followed by double selection with puromycin and neomycin. shRNA was transduced using lentivirus (constitutive KD, puromycin selection) with non-targeting shRNA as control.
Growth protocol J1 mouse embryonic stem cells were routinely cultured in serum and LIF-containing media (+LIF) for self-renewal or without LIF for differentiation (-LIF). Differentiation was carried out for 72 hr or 96 hr as indicated in sample description. Cells were either seeded at high density (1.9e5 cells/cm^2) in a 6-well plate or low density (2.3e4 cells/cm^2) in a 12-well plate or at the densities indicated in sample name in a 24-well plate.
Extracted molecule polyA RNA
Extraction protocol For ChIP-seq, chromatin was sheared from formaldehyde-fixed mouse embryonic stem cells. Protein-DNA complexes of interest were pulled down with magnetic streptaviding beads that were extensively washed in buffer before elution. For RNA-seq, total RNA was isolated using the RNeasy Mini Plus Kit from Qiagen and quantified on a Nanodrop.
For ChIP-seq, libraries were prepared according to the manufacturer's instructions in the NEBNext Ultra™ II DNA Library Prep Kit for Illumina (NEB) and sequenced on a NovaSeq 6000. For RNA-seq, paired-end libraries were prepared according to the manufacturer's instructions in the NEBNext Ultra™ II RNA Library Prep Kit for Illumina (NEB) and sequenced on a NovaSeq 6000. Unpaired libraries were prepared using the NEBNext Ultra™ II RNA Library Prep Kit for Illumina (NEB) and sequenced on a NextSeq500.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description Differentiation in -LIF for 72 hr
Data processing For ChIP-seq, reads from .fastq files were aligned to the mm10 genome using Bowtie 2 version 2.4.2 using multithreading and default settings.
Peaks were then called using MACS2 version 2.2.7.1 using input as control and an effective genome size of 2652783500. Input represents chromatin-associated DNA from mouse embryonic stem cells that were grown, differentiated, fixed, sheared, washed, etc. the same as samples but without dox treatment (meaning no expression of biotinylated transcription factor).
For RNA-seq, reads were pseudoaligned to the mm10 genome using kallisto version 0.46.1 with sequence bias correction enabled as well as bootstrapping (n = 100) in either single or paired-end mode depending on the samples.
Genome_build: mm10
Supplementary_files_format_and_content: peak text files for ChIP-seq, TPM output for RNA-seq
 
Submission date May 30, 2021
Last update date Nov 24, 2021
Contact name Jonghwan Kim
E-mail(s) jonghwankim@mail.utexas.edu
Organization name University of Texas at Austin
Department Molecular Biosciences
Lab Kim Lab
Street address 2506 Speedway
City Austin
State/province TX
ZIP/Postal code 78712
Country USA
 
Platform ID GPL24247
Series (1)
GSE175801 β-catenin, Tcf7l1, and Esrrb mediate seeding density-dependent gene regulation during mouse embryonic stem cell differentiation
Relations
BioSample SAMN19459223
SRA SRX11027856

Supplementary file Size Download File type/resource
GSM5348215_betacateninKD2_lowdensity_r1abundance.tsv.gz 1.7 Mb (ftp)(http) TSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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