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| Status |
Public on Jan 10, 2025 |
| Title |
single-cell multiome datasets (ATAC + Gene Expression), sgRNA enrichment, CRISPRi |
| Sample type |
SRA |
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| Source name |
HAP1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HAP1
strain: dCas9-KRAB stably expressed single clone
treatment: transduced with lentivirus
library type: mRNA
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| Treatment protocol |
Diploid HAP1 cells were isolated from Hoechst (MCE)-stained HAP1 cells by flow sorting. Lentivirus containing dCas9-KRAB-blast and dCas9-VP64-blast were separately used to construct stably-expressing HAP1 cells with diploid cells. The activation and inhibition efficiency of single clones were verified by infection with lentivirus containing sgRNA targeting the TSS of EZH2. The two most efficient clones of each cell line were selected for perturbation.
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| Growth protocol |
HAP1 (Horizon Discovery) cells were cultured in IMDM (ThermoFisher) supplemented with 10% fetal bovine serum in 5% CO2 at 37°C.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Adherent cells were digested with 0.25% trypsin and collected in a 15mL tube containing serum-containing medium, followed by centrifugation and washing of the cell pellet with serum-free basal medium to obtain a single-cell suspension.
The library construction protocol for single-cell multiome datasets (ATAC + Gene Expression)(scMulti) was performed following the manufacturer's instructions using the Chromium Single Cell Multiome ATAC + Gene Expression Reagent Kits. Nuclei are resuspended and transposed with a Transposase to fragment DNA in open chromatin regions and add adapter sequences. The transposed nuclei are then encapsulated into Gel Beads-in-Emulsion (GEMs) with barcoded primers for both ATAC and gene expression. After GEMs are broken, the products are purified and amplified via PCR to generate sufficient material for library construction. The final ATAC and gene expression libraries are constructed by adding sample indices, size selection, and adapter ligation. Sequencing is performed on an Illumina platform with paired-end reads, capturing both ATAC and gene expression data, including barcodes, UMIs, and sample indices. Finally, the enriched sgRNA library and fragmented cDNA were sequenced on an Illumina NovaSeq6000 PE150 platform, following the standard 10x libraries configuration.
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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 |
Library name: scMulti_CRISPRi(sgEnrich)
single-cell multiome datasets (ATAC + Gene Expression), sgRNA enrichment, CRISPRi
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| Data processing |
The single-cell multiome data was processed by Cell Ranger ARC v2.0.2, including aligning RNA reads to the transcriptome, mapping ATAC reads to the genome, quantifying gene expression levels, and identifying accessible chromatin peaks. The output of Cell Ranger ARC was subsequently analyzed using the R package Signac 1.11.0.In the case of sgRNA enrichment data, the enrichment reads were aligned to a reference genome using Cell Ranger v5.0.1, following the same procedure as for standard scRNA-seq data processing. Reads that failed to align to the reference genome were extracted using Samtools. These extracted reads were subsequently mapped to the sequences of the sgRNA library and its reverse complement, utilizing ACCG as the left anchor (located at the end of the U6 promoter) and GTTT as the right anchor. This mapping process allowed for the identification of sgRNA sequences and the determination of their expression levels in each cell. The resulting data, including cell barcodes and sgRNA UMI counts for each perturbed cell, were added to the dataset.
Assembly: hg19
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz; sgRNA enrichment output files: txt.
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| Submission date |
Jan 08, 2025 |
| Last update date |
Jan 10, 2025 |
| Contact name |
Ke Zhao |
| E-mail(s) |
zk8584918@gmail.com
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| Organization name |
Tianjin Medical University
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| Street address |
Qixiangtai Road, No.22
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| City |
Tianjin |
| ZIP/Postal code |
05000 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE286205 |
Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1 [scMulti] |
| GSE286226 |
Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1 |
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| Relations |
| BioSample |
SAMN46167390 |
| SRA |
SRX27292173 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8720911_KRAB_scMulti_sgenrich.txt.gz |
3.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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