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| Status |
Public on Dec 31, 2024 |
| Title |
CRISPRko, sgRNA enrichment scRNA-seq, rep2 |
| Sample type |
SRA |
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| Source name |
HAP1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HAP1
strain: Cas9 stably expressed single clone
treatment: transduced with lentivirus
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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, dCas9-VP64-blast asn Cas9-Blast were separately used to construct stably-expressing HAP1 cells with diploid cells. The activation, inhibition or knock out efficiency of single clones were verified by infection with lentivirus containing sgRNA targeting the TSS of EZH2. The three 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 RNA sequencing (scRNA-seq) was performed following the manufacturer's instructions using the 10x Genomics Chromium Single Cell 3' Library reagents V3. The protocol involved the generation of gel bead-in-emulsion (GEM) using cells resuspended in a master mix along with partitioning oil and gel beads. The GEMs encapsulated individual cells' poly-A RNA, which was retrotranscribed into cDNA containing an Illumina R1 primer sequence, Unique Molecular Identifier (UMI), and the 10x Barcode. The barcoded cDNA was then subjected to various steps, including cleaning up with Silane DynaBeads, PCR amplification, sgRNA enrichment PCR, and fragmentation of the remaining amplified cDNA. Appropriately sized fragments were selected using SPRIselect reagent. The library was then prepared by ligating paired-end constructs with P5 and P7 sequences and a sample index. 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: sgRNA_enrichment_CRISPRko (rep2)
10x Genomics + sgRNA enrichment PCRs
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| Data processing |
Data processing for scRNA-seq and sgRNA enrichment scRNA-seq datasets was performed using the Cell Ranger software v5.0.1, developed by 10x Genomics. The processing steps for both types of data followed similar procedures. For standard scRNA-seq data, the initial steps involved demultiplexing and barcode processing using Cell Ranger. The software assigned cell barcodes and molecular barcodes (UMIs) to individual reads and performed quality control. Gene counting and aggregation were then carried out to generate a gene-cell matrix, quantifying the expression of genes across cells. In the case of sgRNA enrichment scRNA-seq 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 |
Dec 27, 2024 |
| Last update date |
Dec 31, 2024 |
| 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) |
| GSE285461 |
Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1 [scRNA-seq] |
| GSE286226 |
Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1 |
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| Relations |
| BioSample |
SAMN46002057 |
| SRA |
SRX27201822 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8702830_Cas9_rep2_sgenrich_sgRNA_barcode.txt.gz |
2.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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