|
Status |
Public on Nov 23, 2022 |
Title |
2iLIF_24hr_1 |
Sample type |
SRA |
|
|
Source name |
mouse embryonic stem cells
|
Organism |
Mus musculus |
Characteristics |
cell type: mESC treatment: 2i/LIF media, 24 hours after exit from pluripotency
|
Treatment protocol |
For exit from naive pluripotency, serum/LIF+2i-cultured ES cells were seeded at least 24 h before washing with PBS and changing into medium containing a 1:1 mix of glutamine-free DMEM (11960051; Gibco) and Neurobasal medium including N-2 supplement, B-27 supplement, 2-mercaptoethanol and 2 mM l-glutamine at the indicated time before collection (24 or 40 h). 2i/LIF-cultured ES cells were seeded at least 24 h before being washed with PBS and changed into serum-free maintenance medium without 2i or LIF at the indicated time before collection (12, 24 or 40 h).
|
Growth protocol |
ES cells were maintained on gelatin-coated plates in the following media: serum/LIF, serum/LIF+2i or 2i/LIF. Serum/LIF medium contained knockout DMEM (10829018; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Gemini), 0.1 mM 2-mercaptoethanol, 2 mM l-glutamine and 1,000 U ml−1 LIF (Gemini). To generate serum/LIF+2i maintenance medium, serum/LIF medium was supplemented with 3 μM CHIR99021 (Stemgent) and 1 μM PD0325901 (Stemgent) (2i). 2i/LIF medium contained a 1:1 mix of DMEM/F-12 (11320033; Gibco) and Neurobasal medium (21103049; Gibco) including N-2 supplement (17502048; Gibco), B-27 supplement (17504044; Gibco), 2-mercaptoethanol, 2 mM l-glutamine, LIF and 2i. To generate ES cells in the naive ground state of pluripotency, serum/LIF-cultured ES cells were adapted for three passages to serum/LIF+2i medium or 2i/LIF medium. Adapted cells were used for a maximum of nine passages.
|
Extracted molecule |
total RNA |
Extraction protocol |
RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. Libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer's instructions.
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|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
|
|
Data processing |
Quality control and filtering of raw fastq files was performed using fastp. Sequencing reads were aligned to mm10 using STAR. Raw counts were generated using featurecounts. Assembly: mm10 Supplementary files format and content: Processed data files include raw counts for each sample in CSV format.
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|
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Submission date |
Oct 22, 2022 |
Last update date |
Nov 23, 2022 |
Contact name |
Lydia Finley |
E-mail(s) |
finleyl@mskcc.org
|
Organization name |
Memorial Sloan Kettering Cancer Center
|
Department |
Cell Biology Program
|
Street address |
430 E 67th St
|
City |
New York |
State/province |
NY |
ZIP/Postal code |
10065 |
Country |
USA |
|
|
Platform ID |
GPL24247 |
Series (1) |
GSE216356 |
Amino acid intake strategies define pluripotent cell states |
|
Relations |
BioSample |
SAMN31415022 |
SRA |
SRX17995378 |