|
| Status |
Public on Nov 24, 2021 |
| Title |
RNA-dES-3.1e4cellsmL |
| Sample type |
SRA |
| |
|
| Source name |
Mouse embryonic stem cells, differentiated
|
| Organism |
Mus musculus |
| Characteristics |
cell type: J1 mouse embryonic stem cells
treatment: none
|
| 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 NextSeq 500 |
| |
|
| Description |
Grown in -LIF for 96 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 |
GPL19057 |
| Series (1) |
| GSE175801 |
β-catenin, Tcf7l1, and Esrrb mediate seeding density-dependent gene regulation during mouse embryonic stem cell differentiation |
|
| Relations |
| BioSample |
SAMN19459226 |
| SRA |
SRX11027901 |
