GSM5946278: AMI day 3 [N2677]; Mus musculus; RNA-Seq
Loading of Chromium 10x controller, library generation, and quality control: Libraries were generated according to the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 user guide (10x Genomics; manual number, CG000204 Rev B). A twofold excess of cells was loaded into the 10x Genomics Chromium Controller to reach a target number of 10,000 cells per sample. Fragment length distribution of generated cDNAs and libraries was monitored using the Bioanalyzer High Sensitivity DNA kit (Agilent Technologies; catalog number, 5067-4626). Library quantification was performed using the Qubit dsDNA HS-assay kit (Thermo Fisher Scientific; catalog number, Q32854). Sequencing run: Equal molar proportions of generated mRNA expression libraries were pooled, denatured with NaOH, and diluted to 2 pmol/L as outlined in the Denature and Dilute Libraries Guide (Illumina; document number, 15048776 v02). 1.3 mL of denatured pool was sequenced on an Illumina NextSeq 550 sequencer using one High Output Flowcell for 75 cycles and 400 million clusters (NextSeq 500/550 High Output Kit v2.5; catalog number, 20024906). RNA sequencing was performed with the following settings: 28 bp as sequence read 1; 56 bp as sequence read 2; 8 bp as index read 1; no index read 2.