|
| Status |
Public on Jun 07, 2020 |
| Title |
BJ_1 [re-analyzed] |
| Sample type |
SRA |
| |
|
| Source name |
Fibroblast, BJ
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: BJ
cell type: Human foreskin BJ fibroblasts
passage number: passage 8
harvest time: 48 hours post seeding
|
| Growth protocol |
We culture human embryonic stem cells (H1 and H9) in the chemically defined E8 media. Human foreskin BJ fibroblasts (ATCC, CRL-2522) were cultured in fibroblast medium: DMEM, 10% heat-inactivated FBS, 0.1 mM 2-mercaptoethanol, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, 0.1 mM MEM NEAA and 4 ng ml−1 human bFGF.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested with Trizol reagent and stored at -80 °C until use. Total RNA was extracted using the Direct-zolTM Miniprep kit (R2052).
The standard Agilent SureSelect Strand-Specific RNA sequencing protocol was followed. Briefly, the polyA fraction was purified using two rounds of oligo dT-magnetic bead purification. The resulting polyA fraction was fragmented and first strand cDNA was done with random primers in the presence of Actinomycin D using standard techniques. The resulting first stand cDNA was purified using AMPure Beads and second strand cDNA and end repair was done using standard techniques. The blunt ended double stranded cDNA was incubated with exo- E.coli DNA polymerase to add an adenosine to the ends for T/A cloning of the adaptors. PCR amplification was done in the presence of uracil DNA glycosylase to generate strand specificity. A second round of PCR amplification was done to add unique 6-bp barcodes to each sample and add sequences necessary for flow cell attachment. The resulting mRNA libraries were quantitated using qPCR following the manufacturer's instructions (Kapa Biosystems, Woburn MA).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
BJ_48h_CACTTC_L004
This is duplicated sample record of GSM1632430 for the convenient retrieval of the complete raw data from SRA.
|
| Data processing |
Initial QC was performed with FastQC and low-quality bases were removed with Trim Galore (v. 0.4.5).
Reads were quasi-mapped and quantified with Salmon (v0.12.0, with `--gencode` and `-k 21` flags for index generation and `-l A, `--gcBias` and `--validateMappings` flags for quasi-mapping).
Gene-level summarization was performed with tximport (v1.10.0). Count normalization and differential expression were performed with DESeq2 (v1.16.1).
Genome_build: hg38
Supplementary_files_format_and_content: Comma delimited file which contain the normalized gene-level counts for each biological sample.
|
| |
|
| Submission date |
Sep 30, 2019 |
| Last update date |
May 17, 2022 |
| Contact name |
Kejin Hu |
| E-mail(s) |
kejinhu@uab.edu
|
| Phone |
205-934-4700
|
| Organization name |
University of Alabama at Birmingham
|
| Department |
Biochemistry
|
| Lab |
Hu Lab
|
| Street address |
1825 University Boulevard
|
| City |
Birmingham |
| State/province |
AL |
| ZIP/Postal code |
35294 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE138170 |
Profiling of the reprogramome and quantification of fibroblast reprogramming to pluripotency |
|
| Relations |
| Reanalysis of |
GSM1632430 |
| Reanalyzed by |
GSM6164749 |
| BioSample |
SAMN12873201 |
| SRA |
SRX6923624 |
