|
| Status |
Public on May 03, 2017 |
| Title |
Fixed |
| Sample type |
SRA |
| |
|
| Source name |
Cell culture cells, fixed
|
| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
human cell line: Flp-In T-Rex 293 HEK
mouse cell line: NIH-3T3 (ACC 59)
condition: fixed
|
| Treatment protocol |
Cells were grown to 30 to 60% confluence, dissociated with 0.05% bovine trypsin-EDTA (Invitrogen 25300062), quenched with growth medium, and further processed as described previously (Macosko et al. 2015 Cell; Online-Dropseq-Protocol-v.3.1).
|
| Growth protocol |
Cells were grown in DMEM (Invitrogen 61965-026) without antibiotics containing 10% fetal bovine serum, and confirmed to be mycoplasma-free (LookOut Mycoplasma PCR detection kit, Sigma Aldrich).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Methanol-fixation was adapted from Stöckius et al. 2009 (Nat. Methods). To avoid cell clumping, 8 volumes (800 µl) of methanol p. a. (pre-chilled to -20˚C) were added drop-wise, while gently mixing or vortexing the cell suspension.
Macosko et al. 2015 Cell; Online-Dropseq-Protocol-v.3.1.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Drop-seq
Unique lab identifier: NR_CK_028 (ds013_50fix)
|
| Data processing |
Cell and molecular barcodes were extracted from read1 and added as tags using Drop-seq tools v1.12.
Sequenced reads were trimmed for polyA stretches, SMART adaptor sequences and low quality filtering using Drop-seq tools v1.12.
Trimmed sequenced reads were mapped to hg38 and mm10 using STAR_2.4.0j.
Mapped reads were sorted using samtools v1.2 and merged with cell and molecular barcodes using Drop-seq tools v1.12.
Gene annotation tags were added and bead synthesis errors were detected and corrected using Drop-seq tools v1.12.
Cumulative fraction of reads per cell was computed using Drop-seq tools v1.12, inflection point was computed using dropbead v.0.2.
The digital gene expression matrices were computed using Drop-seq tools v1.12.
Genome_build: hg38, mm10
Supplementary_files_format_and_content: *txt: Tab-separated text files containing UMI counts per gene and per cell of each sample.
clusters*.txt files generated using Seurat.
|
| |
|
| Submission date |
Oct 25, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Nikos Karaiskos |
| E-mail(s) |
[email protected]
|
| Organization name |
Max Delbrück Center for Molecular Medicine
|
| Lab |
Systems Biology of Gene Regulatory Elements
|
| Street address |
Hannoversche Str. 28
|
| City |
Berlin |
| ZIP/Postal code |
10115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19415 |
| Series (1) |
| GSE89164 |
Cell fixation and preservation for droplet-based single cell transcriptomics |
|
| Relations |
| BioSample |
SAMN05939264 |
| SRA |
SRX2268042 |
