title: GSM5393362: Bone marrow-derived macrophage - IFNG [scRNA-seq]; Mus musculus; RNA-Seq
library_construction_protocol: 10X Genomics Single Cell 5' Library & Gel Bead Kit Single-cell RNA-seq libraries were prepared using the 10X Single Cell Immune Profiling Solution Kit (v1 Chemistry), according to the manufacturer's instructions. Briefly, FACS sorted cells were washed once with PBS + 0.04% BSA. Following reverse transcription and cell barcoding in droplets, emulsions were broken, and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification (98°C for 45 sec; 14 cycles of 98°C for 20 sec, 67°C for 30 sec, 72°C for 1 min; 72°C for 1 min). For gene expression library construction, 50 ng of amplified cDNA was fragmented, and end-repaired, double-sided size selected with SPRIselect beads, PCR amplified with sample indexing primers (98°C for 45 sec; 14 cycles of 98°C for 20 sec, 54°C for 30 sec, 72°C for 20 sec; 72°C for 1 min), and double-sided size selected with SPRIselect beads. Single-cell RNA libraries were sequenced on an Illumina HiSeq 4000 to a minimum sequencing depth of 25,000 reads/cell using the read lengths 28bp Read1, 8bp i7 Index, 91bp Read2. 