To generate single-cell gel beads in emulsion, single cell suspensions were loaded onto 10X Genomics Single Cell 3′ Chips together with the reverse transcription master mix following the manufacturer's protocol for the Chromium Single Cell 3′ v2 Library (PN-120233, 10X Genomics, Pleasanton, CA). For each sample, sequencing libraries were generated with unique sample indices (SI), assessed by gel electrophoresis (Agilent D1000 ScreenTape Assay, Santa Clara, CA) and quantified with qPCR (Illumina KAPA Library Quantification Kit, Roche, Pleasanton, CA). Following pooling and normalisation to 4 nM, libraries were denatured and diluted to 1.6 pM for loading onto the sequencer. Libraries were sequenced on an Illumina NextSeq 500 (NextSeq Control Software v2.2.0/Real Time Analysis v2.4.11) using NextSeq 500/550 High Output Kit v2.5 (150 Cycles) (Catalogue No. 20024907, Illumina, San Diego, CA) as follows: 26 bp (Read 1), 8 bp (i7 Index), and 98 bp (Read 2).
Chromium single cell 3 v2 library
Manufactured by 10x Genomics
The Chromium Single Cell 3' v2 Library is a lab equipment product designed for single-cell RNA sequencing. It provides a platform for high-throughput analysis of gene expression profiles at the individual cell level.
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Lab products found in correlation
Nextseq 500, by Illumina (1 mentions)
Nextseq 500 550 high output kit v2, by Illumina (1 mentions)
Kapa library quantification kit, by Illumina (1 mentions)
Single cell 3 chip, by 10x Genomics (1 mentions)
D1000 screentape assay, by Illumina (1 mentions)
Chromium system, by 10x Genomics (1 mentions)
Crop seq, by Addgene (1 mentions)
2 protocols using chromium single cell 3 v2 library
1
Single-Cell Transcriptome Sequencing Protocol
2
CRISPR Screening of Trabecular Meshwork Cells
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Comprised of cultured human trabecular meshwork cells (TMWCs) that had been transfected with a CROP-seq (Addgene: 99248) guide RNA (gRNA) pool targeting 128 loci, with the guides targeted to be inserted in the 3′ end of the gene and thus detectable from short-read sequence data. TMWCs were plated in T75 flasks and transfected with a pooled single guide RNA (sgRNA) library lentivirus containing sgRNA for 128 targets, 10 of which were control genes. Cells were harvested 7 days after virus transduction and were FACS sorted for EGFP-positive and viable cells (propidium iodide-negative cells) before applying to the Chromium System (10× Genomics) single cell RNA-sequencing workflow. Single-cell suspensions were used to generate a Chromium library using the Chromium Single Cell 3′ v2 Library (10x Genomics; PC-120237). The estimated number of cells in each well in the Chromium chip was optimized to capture ∼10 000 cells.
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