alias: GSM2733968
title: GSM2733968: PBS_Rep1_P1.S5; Mus musculus; RNA-Seq
library_construction_protocol: After lung dissociation using the GentleMACS lung dissociation kit (Miltenyi Biotec), single cell suspensions were incubated with CD90.2 MicroBeads (Miltenyi Biotec) on ice and enriched for CD90.2+ cells by magnetic separation using LS columns according to the manufacturer’s protocol. CD90.2+ lung cells were then stained on ice with antibodies for sorting. Innate lymphoid cells were defined as 7AAD- CD45+ CD90.2+ CD127+ Lineage (CD11b, CD11c, CD19, NK1.1, CD3ε, CD4, CD8a, TCRβ, TCRγδ)- cells and sorted on a BD FACS Aria (BD Biosciences). For droplet-based scRNA-seq, sorted innate lymphoid cells were encapsulated into droplets, and libraries were prepared using the ChromiumTM Single Cell 3’ Reagent Kits v2 according to manufacturer’s protocol (10x Genomics). The generated scRNA-seq libraries were sequenced using a 75 cycle Nextseq 500 high output V2 kit. For full-length scRNA-seq, single ILCs were sorted into 96-well plates containing 5ul TCL Buffer (QIAGEN) with 1% 2-Mercaptoethanol, centrifuged and frozen at -80°C. SMART-Seq2 protocol was carried out as previously described by Picelli et al. with minor modifications in the reverse transcription step. cDNA was amplified with 22 cycles and tagmented with one-eighth of the standard Illumina NexteraXT reaction volume. Single-cell libraries were pooled and paired-end sequenced (38bp x 2) with a 75 cycle Nextseq 500 high output V2 kit.
identifiers.namespace: GEO
identifiers.id: GSM2733968