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Sample GSM6736637 Query DataSets for GSM6736637
Status Public on Apr 30, 2023
Title human PBMC control
Sample type SRA
 
Source name blood
Organism Homo sapiens
Characteristics cell type: PBMC isolated from fresh blood
tissue: blood
age: anonymous blood donor
Extracted molecule polyA RNA
Extraction protocol For human PBMC control, scRNA was extracted from 10,000 human PBMCs, 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. 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. For the human PBMC 3x loading sample, following modifications were brought to the human PBMC control sample prep: the number of input cells is 30 000, all reagents quantity were doubled, PCR was made in 16 split tubes.
For each sample, barcoded cDNA of 2 PCR tubes were pooled then purified with 0.6X SPRIselect to remove short fragments. Sequencing libraries were prepared with illumina's Nextera XT kit for the purified pooled 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.
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description Scipio bioscience. All provided R1 and R2 FASTQ files are already demultiplexed by sample index.
Data processing All provided R1 and R2 FASTQ files are already demultiplexed by sample index.
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 to quality controls and count matrices.
Detection of cell-associated barcodes was done for each sample by applying the distance-based knee method of UMI-tools based on the first 25 millions input raw reads of the R1 FASTQ file of the sample "10Ka-XT" (human PBMC control sample) and based on the first 37,5 millions input raw reads of the R1 FASTQ file of the sample "30Ka-opt-XT" (human PBMC 3x loading).
Raw read downsampling was perfomed for each sample by randomly subsampling raw reads from the FASTQ files such that the mean number of raw reads belonging to cell-associated barcodes was 20,000.
The provided processed data is composed of one count matrix per sample, computed after raw read downsampling, such that for each sample the same sequencing depth is simulated at 20,000 mean number of raw reads per cell.
Assembly: The reference genome sequences and annotations are based on Ensembl release v99 for human species (GRCh38). Unique Genome Name in Cytonaut v6.7.0: Human_Scipio_2022_A; Species: Homo_sapiens; Genome Source: ensembl; Genome Version: GRCh38; Genome Link: ftp.ensembl.org//pub/release-99/fasta/homo_sapiens/dna/; Annotation Source: ensembl; Annotation Version: 99; Annotation Link: ftp.ensembl.org//pub/release-99/gtf/homo_sapiens/
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)
 
Submission date Nov 17, 2022
Last update date May 01, 2023
Contact name Stuart EDELSTEIN
Organization name Scipio bioscience
Street address 29 rue du Faubourg Saint Jacques, Pépinière Paris Santé Cochin
City Paris
State/province Île-de-France
ZIP/Postal code 75014
Country France
 
Platform ID GPL24676
Series (1)
GSE218212 RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding [scRNA-seq_ScaleUp_Scipio]
Relations
BioSample SAMN31773287
SRA SRX18295237

Supplementary file Size Download File type/resource
GSM6736637_10Ka-XT_threshold_20000_rep_1_ds_count_matrix.tsv.gz 1.1 Mb (ftp)(http) TSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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