title: GSM8519449: RORc+_Cells_Experiment_cDNA; Mus musculus; RNA-Seq
library_name: GSM8519449
library_construction_protocol: GFP+ Immune cells were isolated from 6 months old DEN-injected mice. Immune cells were isolated from 5 IKK RORc Het mice (cells were pooled after isolation). Livers were cut to small pieces in 5 ml liver digest medium, squashed with a syringe piston, and incubated at 37⁰c for 30 min. PBS was added and cells were centrifuged at 350g for 5 min at 4⁰c. Suspension was passed through a 70 µm mesh filter. Two centrifugations at 30g for 5 min at 4⁰c were performed to reduce hepatocytes content. Supernatant was then centrifuged at 350g at 4⁰c to precipitate the immune cells, and the pellet was resuspended in 6ml of 35% Percoll and gently placed on top of 3ml of 75% Percoll. Cells were centrifuged for 20 min at 900g without brake, and the ring of immune cells between the 2 Percoll concentrations was taken out into PBS and centrifuged for 8 min at 350g. Red blood cell lysis was performed for 2 min at RT and cells were resuspended in PBS+2%FBS (FACS buffer). Cells were counted using Trypan blue.  GFP+ cells were sorted using a BD FACSAria™ III sorter. Sorting was performed using a 85-micron nozzle and threshold was kept in the range of 3000 events/second. Cells were sorted into Eppendorf tubes containing 1ml FACS buffer, and then centrifuged without brake. Cells were counted and brought to a concentration of 1000 cells/µl. Cells were loaded onto a Chromium single-cell 3′ v2 chip (10x Genomics) and libraries were prepared according to the manufacturer's protocol. Libraries were sequenced by Illumina HiSeq 2500 and the reads were mapped to mm10 library (ensembl v98).
identifiers.namespace: GEO
identifiers.id: GSM8519449_r1