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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 29, 2023 |
| Title |
HEKNIH, control |
| Sample type |
SRA |
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| Source name |
NIH; HEK
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell type: NIH; HEK
strain: NIH 3T3 (ECACC 93061524); HEK293 (ACC 305, DSMZ)
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For each sample, scRNA was extracted from a mix of NIH and HEK cells (50%-50%; 10,000 cell input), according to the RevGel-Seq protocol instructions. Briefly, Barcode beads and cells are attached via a bifunctional chemical linker. These tandems are dispersed in an hydrogel in the liquid state and immobilized upon gelation. For the sample HEKNIH freezer and HEKNIH dry ice, the hydrogel with cell-bead tandems is frozen right after immersion to lysis buffer. This freezeing was done in -80°C freezer for the sample HEKNIH freezer, on dry ice for the sample HEKNIH dry ice. The day after, the frozen sample was thawed at 25°C for 15 min to resume cel lyeis step. Following cell lysis, polyA+ RNA molecules are captured on the barcoded beads via polyA-polyT interactions. Barcoded beads (and associated RNA molecules) are recovered following degelation, and reverse transcription and subsequent second strand synthesis with random primer are performed. Each sample is then split in 8 subfractions and PCR-amplified to obtain cell barocded cDNA.
For each of the 3 samples, cDNA from 3 subfractions of PCR were individually purified with SPRIselect to remove short fragments. Sequencing libraries were prepared with illumina's Nextera XT kit for each of the 3 purified barcoded cDNA according to manufacturer's instructions with the following adaptations: 600pg of the cDNA was subjected to tagmentation reaction. The tagmented DNA was amplified by 12 cycles PCR. During this PCR, DNA fragments with cell barcode sequence at one end are selectively amplified by using a primer pair of custom P5-read1 primer to the original barcode end and Nextera N7xx P7 index primer to the tagmented end. Paired end sequencing was done.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Scipio bioscience. All provided R1 and R2 FASTQ files are already demultiplexed by sample index.
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| Data processing |
All provided R1 and R2 FASTQ files are already demultiplexed by sample index.
For each sample subfraction (3 subfractions per sample were sequenced), one couple of R1 and R2 FASTQ files was generated;
Data pre-processing (read quality control, extraction, alignment, deduplication and assignment) was performed using Cytonaut v6.7.0 (https://cytonaut-scipio.bio) from raw FASTQ files of each sample subfraction to quality controls and count matrices.
Detection of cell-associated barcodes was done for each sample subfraction by applying the distance-based knee method of UMI-tools based on 12,5 millions input raw reads randomly sampled from the concatenated R1 FASTQ file of the sample subfraction.
Raw read downsampling was perfomed for each sample subfraction by randomly subsampling raw reads from the FASTQ files such that the mean number of raw reads belonging to cell-associated barcodes was 50,000.
The provided processed data is composed of one count matrix per sample subfraction (thus 3 count matrices per sample), computed after raw read downsampling, such that for each sample the same sequencing depth is simulated at 50,000 mean number of raw reads per cell.
Assembly: The reference genome sequences and annotations are based on Ensembl release v99 for human species (GRCh38) and mouse species (GRCm38). Unique Genome Name in Cytonaut v6.7.0: Human_Mouse_Scipio_2022_A; Species: Homo_sapiens & Mus_musculus; Genome Source: ensembl & ensembl; Genome Version: GRCh38 & GRCm38; Genome Link: ftp.ensembl.org//pub/release-99/fasta/homo_sapiens/dna/ & ftp.ensembl.org//pub/release-99/fasta/mus_musculus/dna/; Annotation Source: ensembl & ensembl; Annotation Version: 99 & 99; Annotation Link: ftp.ensembl.org//pub/release-99/gtf/homo_sapiens/ & ftp.ensembl.org//pub/release-99/gtf/mus_musculus/.
Supplementary files format and content: Tab Separated Values (TSV format) including all count matrix information (genes in first column, then one column per cell-associated barcode)
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| Submission date |
Nov 17, 2022 |
| Last update date |
Mar 29, 2023 |
| Contact name |
Stuart EDELSTEIN |
| Organization name |
Scipio bioscience
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| Street address |
29 rue du Faubourg Saint Jacques, Pépinière Paris Santé Cochin
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| City |
Paris |
| State/province |
Île-de-France |
| ZIP/Postal code |
75014 |
| Country |
France |
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| Platform ID |
GPL25526 |
| Series (2) |
| GSE218209 |
RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding [scRNA-seq_stopping_Scipio] |
| GSE218213 |
RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding |
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| Relations |
| BioSample |
SAMN31773283 |
| SRA |
SRX18295241 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6736630_Ctrl-1_1_S16_threshold_50000_rep_1_ds_count_matrix.tsv.gz |
3.0 Mb |
(ftp)(http) |
TSV |
| GSM6736630_Ctrl-2_1_S17_threshold_50000_rep_1_ds_count_matrix.tsv.gz |
2.9 Mb |
(ftp)(http) |
TSV |
| GSM6736630_Ctrl-2_4_S18_threshold_50000_rep_1_ds_count_matrix.tsv.gz |
3.2 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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