|
| Status |
Public on Jul 15, 2016 |
| Title |
Male Naive ES - WIN1 (XY) |
| Sample type |
SRA |
| |
|
| Source name |
human ES
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: WIN1
es state: naive
medium: 4i/L/A
|
| Growth protocol |
Conventional (primed) human ESC lines were maintained on mitomycin C inactivated mouse embryonic fibroblast (MEF) feeders in human embryonic stem cell medium (hESM) and passaged mechanically using a drawn Pasteur pipette or enzymatically by treatment for 20 minutes with 1 mg/mL Collagenase Type IV (Gibco) followed by sequential sedimentation steps in hESM to remove single cells. Naive human ESCs were cultured on mitomycin C-inactivated MEF feeder cells, and were passaged by a brief PBS wash followed by single-cell dissociation using 3-5 minute treatment with Accutase (Gibco) and centrifugation in fibroblast medium. For conversion of pre-existing primed human ESC lines, we seeded 2 x 105 trypsinized single cells on a MEF feeder layer in hESM supplemented with ROCK inhibitor Y-27632 (Stemgent, 10 μM). 2 days later medium was switched to 5i/L/A or 4i/L/A (no IM12)-containing naive human ESC medium. Following an initial wave of cell death, naive colonies appeared within 10 days and were expanded polyclonally using Accutase (Gibco) on a MEF feeder layer. Naive cells generated with DOX-inducible KLF2 and NANOG transgenes (Theunissen et al., 2014) were maintained in 2i/L/DOX with optional inclusion of 10 μM ROCK inhibitor Y-27632. To adapt DOX-dependent naive cells to transgene-free culture conditions, 1 x 105 single cells were seeded on a MEF feeder layer in 2i/L/DOX and DOX was withdrawn the next day. For re-priming, semi-confluent cultures of naive cells were switched to hESM with ROCK inhibitor Y-27632 (10 μM) and passaged with Collagenase on MEFs. Neuronal precursor cells (NPCs) were derived from re-primed cells using a published differentiation protocol (Cohen et al., 2007). To culture naive human cells described by Gafni et al. (2013), we used the KSR-based version of the NHSM protocol. Tissue culture media were filtered using a low protein-binding binding 0.22 μm filter (Corning). All experiments in this paper were performed under physiological oxygen conditions (5% O2, 3% CO2). Detailed media compositions are described in Supplemental Experimental Procedures.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Illumina Truseq Stranded RNA
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Illumina pipeline Casava 1.82
Reads were mapped to the human genome (hg19) using TopHat (v2.0.11) in sensitive mode (tophat --library-type fr-firststrand -g 1 --no-novel-juncs --no-novel-indels –b2-sensitive)
Gene counts were generated using HTSeq-count (0.6.1)
Repeats counts were generated using the multiCov tool from the bedtools (2.25) software
Genome_build: hg19
Supplementary_files_format_and_content: read counts on genes (pileup files): htseq-cout output; read counts on repeats: bedtools multiBamCov output
|
| |
|
| Submission date |
Jun 27, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Joseph R Ecker |
| E-mail(s) |
ecker@salk.edu
|
| Phone |
8584534100
|
| Organization name |
HHMI-Salk-Institute
|
| Department |
Genomic Analysis Laboratory
|
| Lab |
Ecker lab
|
| Street address |
10010 North Torrey Pines Road
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE75868 |
Molecular Criteria for Defining the Naive Human Pluripotent State |
| GSE83765 |
RNA-seq of naive and primed ES cells |
|
| Relations |
| BioSample |
SAMN05294425 |
| SRA |
SRX1879687 |
