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| Status |
Public on Dec 01, 2021 |
| Title |
HEK293T_proof-of-concept |
| Sample type |
SRA |
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| Source name |
Cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
treatment: none
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| Treatment protocol |
For the proof of concept experiment, HEK293T cells were grown in a T-75 flask. For the flavopiridol time course experiment, HEK293T cells were seeded onto a 6-well culture plate at a density of 400,000 cells per well in 2 mL of media. For the HDAC inhibitor time course and HDAC inhibitor and dexamethasone co-treatment experiments, A549 cells were seeded onto a 96-well culture plate at a density of 25,000 cells per well in 100 μL of media.
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| Growth protocol |
A549 and HEK293T cells were cultured in DMEM (Gibco) media containing 10% fetal bovine serum (Invitrogen) and 1% penicillin and streptomycin (Gibco) at 37°C with 5% CO2.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
After drug treatment, cells were harvested via trypLE incubation and lysed with ice cold cell lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Extracted nuclei were then labeled with hash oligos by paraformaldehyde fixation with appropriate hash oligos and subjected to sci-RNA-seq (Cao et al. 2017). Detailed protocol is described in the method section of the manuscript.
sci-RNA-seq libraries were prepared by following the protocol described in Cao et al. 2017 and Srivatsan et al. 2020.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
sci-RNA-seq profiles and hash ladder calibration lines of HEK293T cells
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| Data processing |
Illumina RTA software used for basecalling.
The sci-RNA-seq libraries were processed according to the protocol described in Srivatsan et al. First, Illumina reads were demultiplexed using bcl2fastq (v2.20). Custom scripts were used to extract barcodes that match reverse transcription (RT) indices within Levenstein distance cutoff of 2. After RT barcode matching, poly(A) sequences were trimmed using trim-galore, and the trimmed reads were aligned to hg38 using STAR aligner (v2.5.2b) with default settings. The aligned reads were filtered with MAPQ >= 30, deduplicated, and collapsed by unique molecular identifiers (UMIs). The deduplicated reads were then assigned to genes using bedtools intersect function with an annotated human gene model. Knee plots were then generated from UMI counts per cell barcode to filter out true cell barcodes from debris. Thresholds were selected on a per-experiment basis and gene expression profiles from cell barcodes with total UMI counts greater than the knee threshold were used to generate a CDS object for downstream analysis.
The hash IDs were assigned sample labels as described in Srivatsan et al. In summary, reads from hash oligos were extracted from the demultiplexed read files by matching the first 10 bp of read 2 to the hash barcodes with the Levenstein distance of 2 and looking for trailing poly(A) sequences from position 12-16 bp. These reads were deduplicated and collapsed by UMIs, which were then used for sample assignment and hash ladder calibration line construction.
Genome_build: GRCh38
Supplementary_files_format_and_content: _UMI.count.matrix: single cell gene expression data in a sparse matrix format (gene_index | cell_index | gene_count).
Supplementary_files_format_and_content: _cell.annotations: Cell annotation file (cell_barcode | experiment name).
Supplementary_files_format_and_content: _gene.annotations: Gene annotation file (gene_id | gene_short_name).
Supplementary_files_format_and_content: _hashLadderSampleSheet.txt: Sample sheet for hash oligos used for calibration.
Supplementary_files_format_and_content: _hashIDSampleSheet.txt: Sample sheet for hash oligos used for labeling different treatment conditions.
Supplementary_files_format_and_content: _hash-ladder-exp.txt: Sample sheet for hash oligos used for labeling different treatment conditions.
Supplementary_files_format_and_content: _hashID-annotation.txt: Sample sheet for hash oligos used for labeling different treatment conditions.
Supplementary_files_format_and_content: _hashTable.out: Table that contains hash ladder oligo counts for each cell in the experiment.
Supplementary_files_format_and_content: _hashTable-labels-filtered.out: Table that contains hash ID oligo counts for each cell in the experiment.
Supplementary_files_format_and_content: _hashTable_unique_filtered.txt: Table that summarizes hash ladder calibration lines in each cell
Supplementary_files_format_and_content: _cds_conv.rds: Monocle3 cds object constructed with conventional size factor normalization.
Supplementary_files_format_and_content: _cds_hash_ladder.rds: Monocle3 cds object constructed with hash ladder based size factor normalization.
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| Submission date |
Feb 09, 2021 |
| Last update date |
Dec 01, 2021 |
| Contact name |
Hyeon-Jin Kim |
| E-mail(s) |
khj3017@uw.edu
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| Organization name |
University of Washington
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| Department |
Genome Sciences
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| Lab |
Cole Trapnell, Doug Fowler
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| Street address |
3720 15th Ave NE
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE166470 |
Nuclear oligo hashing improves differential analysis of single-cell RNA-seq |
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| Relations |
| BioSample |
SAMN17843392 |
| SRA |
SRX10060654 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5071636_HEK293T_proof-of-concept_UMI.count.matrix.mtx.gz |
21.3 Mb |
(ftp)(http) |
MTX |
| GSM5071636_HEK293T_proof-of-concept_cds.rds.gz |
18.1 Mb |
(ftp)(http) |
RDS |
| GSM5071636_HEK293T_proof-of-concept_cell.annotations.txt.gz |
4.6 Kb |
(ftp)(http) |
TXT |
| GSM5071636_HEK293T_proof-of-concept_gene.annotations.txt.gz |
420.0 Kb |
(ftp)(http) |
TXT |
| GSM5071636_HEK293T_proof-of-concept_hash-ladder-exp.txt.gz |
126 b |
(ftp)(http) |
TXT |
| GSM5071636_HEK293T_proof-of-concept_hashLadderSampleSheet.txt.gz |
142 b |
(ftp)(http) |
TXT |
| GSM5071636_HEK293T_proof-of-concept_hashTable.out.txt.gz |
159.2 Kb |
(ftp)(http) |
TXT |
| GSM5071636_HEK293T_proof-of-concept_hashTable_unique_filtered.txt.gz |
112.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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