|
| Status |
Public on Nov 23, 2022 |
| Title |
2iSL_40hr_2 |
| Sample type |
SRA |
| |
|
| Source name |
mouse embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: mESC
treatment: 2i + serum/LIF media, 40 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.
|
| |
|
| 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.
|
| |
|
| 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 |
SAMN31415011 |
| SRA |
SRX17995389 |
