title: GSM3382153: NRG-HIS_ID305_Day40_CD33+; Homo sapiens; RNA-Seq
library_construction_protocol: Frozen splenocytes from NFA2-HIS/Flt3LG and NRG-HIS mice were thawed and stained with antibodies for anti-human CD33 or CD45. Cell populations that were either anti-human CD33+ or anti-human CD45+ were sorted into RPMI + 10% FBS. Single cells from each sort gate and each animal were loaded onto prepared Seq-Well arrays. Briefly, 10,000 single cells from each sort gate were loaded onto one array containing 86,000 barcoded mRNA capture beads. Cells and beads were co-confined in microwells on the array, and a polycarbonate membrane sealed individual wells to allow for isolated single-cell lysis and transcript hybridization prior to bead recovery for reverse transcription. each library was treated with Exonuclease I to remove excess primers, and PCR amplification with KAPA Hifi PCR Mastermix (Thermo Scientific, Waltham, MA, USA) to generate final cDNA libraries. Libraries were then constructed using the Nextera XT DNA tagmentation method (Illumina, San Diego, CA, USA). Tagmented and amplified libraries were subsequently purified and sequenced using an Illumina 75 cycle NextSeq500/550v2 kit (paired end reads, custom Read 1 primer, see Gierahn et al Nature Methods 2017, 20bp Cell Barcode and UMI, 50 bp DGE). scRNA-seq - SeqWell (Gierahn et al. Nature Methods. 2017)
identifiers.namespace: GEO
identifiers.id: GSM3382153