|
| Status |
Public on Jul 16, 2015 |
| Title |
P3_E6 |
| Sample type |
SRA |
| |
|
| Source name |
hiF-T cells
|
| Organism |
Homo sapiens |
| Characteristics |
reprogramming time point (day): 5
treatment: 1 uM ROCK inhibitor
|
| Growth protocol |
Refer to the section Cell Culture and Reprogramming in the Supplemental Experimental Procedures of the related manuscript.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA, including the small RNA fraction, was obtained by organic extraction followed by miRNeasy purification (Qiagen).
The 3’-DGE libraries were prepared from 20ng of total RNA according to the Single Cell RNA Barcoding and Sequencing method originally developed for single cell RNA-seq (SCRB-seq (Soumillon et al., 2014) and adapted to extracted total RNA. Briefly, Poly(A)+ mRNA from extracted total RNA are converted to cDNA decorated with universal adapters, sample-specific barcodes and unique molecular identifiers (UMIs) using a template-switching reverse transcriptase. Decorated cDNA from multiple samples are then pooled, amplified and prepared for multiplexed sequencing using a modified transposon-based fragmentation approach that enriches for 3’ ends and preserves strand information.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
ROCKi5_R2
|
| Data processing |
Paired reads were used for analysis if all sixteen bases of the first read had quality scores of at least 10 and the first six bases corresponded exactly to a designed well-barcode. All second sequence reads were aligned to a reference database consisting of all human RefSeq mRNA sequences (hg19) and the ERCC RNA spike-in reference sequences using bwa version 0.7.4 4 with non-default parameter “-l 24”. Reads mapped to multiple genes were excluded from further analysis. Digital gene expression (DGE) profiles were then generated by counting, for each microplate well and RefSeq gene, the number of unique molecular identifiers (UMIs) associated with alignments to that gene in that well.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text file includes UMI values for each sample.
|
| |
|
| Submission date |
May 29, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Shannan J Ho Sui |
| E-mail(s) |
shosui@hsph.harvard.edu
|
| Organization name |
Harvard T.H. han Scholol of Public Health
|
| Department |
Biostatistics
|
| Street address |
655 Huntington Ave, Building 2, Rm 437A
|
| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE62777 |
Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency |
| GSE69351 |
Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [SCRB-Seq] |
|
| Relations |
| BioSample |
SAMN03743519 |
| SRA |
SRX1042168 |
