|
| Status |
Public on May 01, 2016 |
| Title |
P3_D12 - BT20_1250_day2 |
| Sample type |
SRA |
| |
|
| Source name |
BT20
|
| Organism |
Homo sapiens |
| Characteristics |
growth time (days): 3
seeding density (cell/well): 1250
|
| Treatment protocol |
No treatment
|
| Growth protocol |
All cells lines were obtained from the ATCC and grown according to their recommendations.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy mini kit (Qiagen).
The 3’-DGE libraries were prepared from 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 |
BT20 cells seeded at 1250 cells per well (384-well plate), harvasted 3 days after seeding
|
| 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-separated file with columns as samples (labelled as P3_*) and genes as rows with UMI count for each gene/sample pair.
|
| |
|
| Submission date |
Apr 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Marc Hafner |
| E-mail(s) |
Marc_Hafner@hms.harvard.edu
|
| Organization name |
Harvard Medical School
|
| Department |
Systems Biology
|
| Lab |
Sorger Lab
|
| Street address |
WAB 438, 200 Longwood Av.
|
| City |
Boston |
| State/province |
MASSACHUSETTS |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE80297 |
mRNA expression of breast cancer cell lines across different densities [SCRB-Seq] |
|
| Relations |
| Reanalyzed by |
GSE123917 |
| BioSample |
SAMN04856847 |
| SRA |
SRX1705402 |
