title: GSM6205415: 20CQ_Experiment2; Mus musculus; OTHER
library_construction_protocol: Single-cell suspensions were made from the spleen. Processing of cells and flow cytometry staining were performed on ice. Briefly, cells were lysed (ACK red cell lysis buffer, Gibco), passed through a 70μm cell strainer (Fisher Scientific), washed, and resuspended in ice cold phenol red-free DMEM (Gibco) with 2% (vol/vol) Fetal Calf Serum FACS buffer at 4 x 108 cells/mL. For surface staining, cells were incubated for 5 mins with 2% (vol/vol) normal mouse and normal rat serum (Jackson ImmunoResearch Laboratories), prior to staining with a 2X master mix containing fluorophore-labeled antibodies at pre-titrated concentrations in Brilliant Violet Staining Buffer (BD Biosciences), and added to suspended cells at a final staining concentration of 2 x 108 cells/mL for 30-45 min. Cells were washed and resuspended in FACS buffer before flow cytometry analysis and sorting Library preparation was carried out using a custom scRNA-seq strategy described in detail in (Shuparksi et al., 2021 STAR Protocols). In brief, Single cells were sorted into 384 well plates with each well containing the necessary reagents for reverse transcription (RT). Including polyA targeting oligoDT primers with unique molecular identifiers and cell barcodes. Negative control wells without sorted cells were included to enable assessment of potential contamination. Following the completion of the sort an RT reaction was performed (42˚C for 50 mins then 80˚C for 10 mins) and wells from the same plate were pooled into a single PCR tube. To remove excess oligoDT primer, cDNA libraries were treated with ExoSAP-IT Express according to manufacturer's instructions (Affymetrix). Libraries were then volume reduced via a 0.8x ratio of AMpure XP SPRIselect magnetic beads (BeckmandCoulter). Ensuring separation between plates to preserve barcode fidelity, two nested PCR reactions were performed utilizing external and internal mixed primer sets targeting ~500 gene transcript species of interest. A final PCR reaction was carried out to allow for the ligation of adapter sequences enabling Illumina flow cell binding. Final cDNA libraries containing the amplified targeted mRNA species were quantified using a Qubit 4 Fluorometer (Invitrogen Technologies) and 2100 BioAnalyzer (Agilent). Multiplexed plates were then sequenced on an Illumina NextSeq 500 (Read 1: 19 cycles; Index Read 1: 6 cycles; Read 2: 67 cycles)