title: GSM8834325: Thymic rosette APC; Mus musculus; RNA-Seq
library_name: GSM8834325
library_construction_protocol: Three thymi from 6-week old C57Bl/6 female mice were harvested and minced with scissors. The tissue pieces were washed in RPMI + 5mM EDTA with gentle rocking for 15 minutes to release free thymocytes. The fragments were then digested with 0.5mg/mL Collagenase D, 4μg/mL DNase I and 2% FCS in RPMI at room temperature with magnetic stirring three times for 15 min each to release rosettes. The resulting digest containing rosettes (and all subsequent washes) were centrifuged at 100xg for 7 minutes at 4°C and resuspended in RPMI. This suspension was overlaid on newborn calf serum and left to settle at room temperature. After 15-20 min of incubation, the cells within the RPMI layer were discarded. The serum layer, now enriched for rosettes were collected and washed with PBS, was resuspended in RPMI for subsequent sedimentations. A total 4 sedimentations were performed to enrich for rosettes. For flow cytometric sorting, rosettes were first dissociated into single cells using 0.25% v/v trypsin for one minute at 37°C. 3' Gene Expression library was constructed using the standard Chromium Single Cell 3' reagent kit (v3 chemistry) (10x Genomics). Briefly, Gel beads-in-emulsion (GEM) were generated, containing a single cell and a barcoded gel bead. Following GEM formation, the gel bead was dissolved to release primers, and the cell was lysed. The primers capture polyadenylated mRNA using a 30nt poly(dT) sequence, and they also contain a 16nt 10x barcode, a 12nt unique molecular identifier, and Illumina TruSeq Read 1 primer sequence. cDNA is then reverse transcribed from mRNA and amplified using PCR. P5 and P7 sequences were also attached to size-optimised cDNA. The library was then sequenced on an Illumina NextSeq 500.
identifiers.namespace: GEO
identifiers.id: GSM8834325_r1