GSM4504503: Anti-Ang2 no IR; Mus musculus; RNA-Seq
In order to obtain single cell solution, incubated in Hanks' balanced salt solution (HBSS, 14175-053, Gibco) supplemented with 1 mg/mL collagenase type I (LS004196, Worthington), 1 mg/mL collagenase H (11074032001, Roche), 4 mg/mL dispase II (04942078001, Sigma) and 1000 U/mL benzonase (sc-202391, ChemCruz) for 30 min at +37°C with gentle shaking, followed by 15 min incubation with Trypsin-EDTA (25200056, Thermo Fisher Scientific) and red blood cell lysis buffer (ACK Lysing Buffer, A1049201, Gibco) for 10 minutes in RT. Cells were transferred into 0.04% BSA-HBSS for analysis. Single cells were analyzed using the 10x Chromium Single-Cell 3′RNA-sequencing system (10x Genomics, Pleasanton, CA, USA) with the Reagent Kit v3 as instructed by the manufacturer. Briefly, the cells were first loaded into Chromium Single-Cell Chip v3 (10x Genomics, Pleasanton, CA, USA), followed by generation of Gel beads in EMulsion (GEM) aiming at 10 000 cell captures per sample. This was followed by cDNA purification, amplification (12 cycles), and library construction (sample index PCR 14 cycles) as instructed by the manufacturer. The sample libraries were sequenced on an Illumina NovaSeq 6000 sequencer using read lengths of 26bp (Read 1), 8bp (i7 Index), 0 bp (i5 Index) and 91bp (Read 2). Chromium single-cell 3' RNA-sequencing barcodes and read data were demultiplexed and converted to FASTQ files, and aligned reads and gene-cell matrices were generated using Cell Ranger v 2.1.1 mkfastq and count pipelines (10x Genomics, Pleasanton, CA, USA). Reads were aligned to mouse reference genome mm10.
GEO Accession
GSM4504503