In total, the cell viability of all samples is above 90%, 10,000 cells per sample were loaded into a Chromium Single-Cell 3′ Chip Kit v2 and processed using a Chromium single cell 3′ Reagent Kits (v2 and v3) (10× Genomics) according to the manufacturer’s instructions. Libraries were constructed using the Single 3′ Library and Gel Bead Kit v2 (PN-120237) and Chromium i7 Multiplex Kit v2 (PN-120236).
Chromium single cell 3 chip kit v2
Manufactured by 10x Genomics
The Chromium Single Cell 3' Chip kit v2 is a lab equipment product from 10x Genomics. It is designed for single-cell RNA sequencing applications, allowing for the capture and analysis of transcriptomes from individual cells.
Automatically generated - may contain errors
Lab products found in correlation
Chromium i7 multiplex kit, by 10x Genomics (3 mentions)
Chromium single cell 3 library and gel bead kit v2, by 10x Genomics (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Chromium single cell 3 library gel bead kit v2, by 10x Genomics (2 mentions)
Chromium single cell 3 reagent kits v2 and v3, by 10x Genomics (1 mentions)
Deoxyribonuclease 1 dnase 1, by Merck Group (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Single cell 3 library and gel bead kit v2, by 10x Genomics (1 mentions)
Purelink rna mini kit columns, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
6 protocols using chromium single cell 3 chip kit v2
1
Single-Cell RNA Sequencing of Cell Cultures
2
Single-cell RNA-seq on THY1+ cells
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The MACS-sorted THY1+ cells were used for loading onto the Chromium Single Cell 3′ Chip kit v2 (10x Genomics, PN-120236) according to the instructions. Cell capturing and library preparation was performed following the kit instructions of the Chromium Single Cell 3’ v2 Library and Gel Bead Kit (10x Genomics, PN-120237). In brief, 5000 cells were targeted for capture, and after cDNA synthesis, 10–12 cycles were used for library amplification. The libraries were then size-selected, pooled, and sequenced on a Novaseq 6000 (Illumina). Shallow sequencing was performed to access the library quality and to adjust the subsequent sequencing depth based on the capture rate and the detected unique molecular indices (UMI).
3
Single-cell RNA-seq of mouse testes
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Testes from WT, W, W3, and W5 mice were minced and digested with collagenase type IV (1 mg/ml; Sigma-Aldrich) and deoxyribonuclease I (DNase I; 500 μg/ml; Sigma-Aldrich) at 37°C for 15 min. The cell suspension was pipetted up and down once every 5 min, and the digestion process was stopped using Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS). Under each condition, testicular single-cell suspensions from three biological replicates were mixed and filtered through a 40-μm nylon mesh. After centrifugation, the cells were resuspended in 0.04% BSA in phosphate-buffered saline (PBS) for loading onto the Chromium Single Cell 3′ Chip kit v2 (10x Genomics, PN-120236). Cell capture and library preparation were performed following the instructions of Chromium Single Cell 3′ v2 Library and Gel Bead Kit (10x Genomics, PN-120237). Briefly, 10,000 cells were targeted for capture per sample, and after cDNA synthesis, 10 to 12 cycles were used for library amplification. The libraries were then size-selected, pooled, and sequenced on a NovaSeq 6000 (Illumina). Shallow sequencing was performed to assess library quality and adjust the subsequent sequencing depth based on the capture rate and detected unique molecular indices (UMIs).
4
High-throughput single-cell RNA sequencing
5
High-throughput single-cell RNA sequencing
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For MARS-seq scRNA protocol, single cells were sorted into 384-well plates containing 2 μl of barcoded RT primers in concentration of 8 nM in each well. Downstream library preparation was done according to Jaitin et al. 2014, and by using randomized UMI sequence of 8 base-pairs (allowing maximal count of ~65k UMIs per gene per well). For NCI-H1299 scRNA, we used data of ‘5 10x libraries described in Brocks et al. 201923 (link). HCT116 WT and HCT116 DKO scRNA libraries based on 10x Genomic platform, were generated using Chromium™ Single Cell 3’ Library & Gel Bead Kit v2 (PN-120237), Chromium Single cell 3’ Chip Kit V2 (PN-120236) and Chromium i7 Multiplex Kit (PM-120262), following the manufacturer’s instructions (10x Genomics®, Inc.). Two samples of 5,000 cells were loaded per each, HCT116 PAR and HCT116 DKO-033 libraries were sequenced paired-end 150 bp on Nextseq 500 to mean depth of 68,257 and 45,259 reads per cell for PAR and DKO cells, respectively.
6
Bulk and Single-cell RNA-seq Protocols
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Single-cell RNA-sequencing (scRNA-Seq) was performed on the 10x Genomics platform with Chromium Single Cell 3′ Chip Kit v2 (10x Genomics cat#1000009). Libraries were constructed using the Single Cell 3′ Library and Gel Bead Kit v2 (10x Genomics cat#120237) and Chromium i7 Multiplex Kit 10x Genomics cat#120262). Two single-cell libraries were pooled and sequenced per HiSeq 2500 125-base PET lane.
Conventional (bulk) RNA-sequencing was performed on unfractionated cell suspension or snap frozen whole tissue material. Total RNA was isolated with TRIzol reagent followed by purification over PureLink RNA Mini Kit columns (Invitrogen cat#12183018 A). RNA-seq was performed using a polyA-enriched strand-specific library construction protocol56 (link) and paired-end 75 bp sequencing on an Illumina HiSeq 2500 instrument.
Conventional (bulk) RNA-sequencing was performed on unfractionated cell suspension or snap frozen whole tissue material. Total RNA was isolated with TRIzol reagent followed by purification over PureLink RNA Mini Kit columns (Invitrogen cat#12183018 A). RNA-seq was performed using a polyA-enriched strand-specific library construction protocol56 (link) and paired-end 75 bp sequencing on an Illumina HiSeq 2500 instrument.
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