title: GSM8549737: GT8-IL-21-NIC; Mus musculus; RNA-Seq
library_construction_protocol: Antigen-specific germinal center B cells were sorted and subsequently uniquely barcoded using the 10× Genomics Chromium single-cell platform, and complementary DNA (cDNA) libraries were prepared for Next Generation Sequencing according to the manufacturer's protocol (Chromium NextGem Single Cell 3' V2 Library and Gel Bead Kit, 10× Genomics, USA). Cell suspensions of each sample, reverse transcription master mix, and partitioning oil were loaded on a single-cell “G” chip with a targeted cell output of 10,000 cells per library and then run on the Chromium Controller. A total of 4 lanes were used on the G chip, 1 lane/sample. Reverse transcription was performed within the droplets at 53°C for 45 min and newly synthesized cDNA was amplified for 11 cycles on a Veriti Thermal Cycler (Thermofisher, USA). cDNA size selection was performed using SPRIselect beads (Beckman Coulter, USA) following manufacturer's protocol. cDNA was analyzed on an Agilent Tapestation High Sensitivity D5000 (Agilent, USA) for qualitative and quantitative control purposes. cDNA was then used to make libraries according to manufacturer's protocol (Chromium Next GEM Single Cell 3' Reagent Kit v3.1). cDNA was fragmented using the proprietary fragmentation enzyme blend for 5 min at 32°C, followed by end-repair and A-tailing at 65°C for 30 min. cDNA was double-sided size selected using SPRIselect beads. Sequencing adaptors were ligated to the cDNA at 20°C for 15 min. and after a round of post-ligation SPRIselect bead clean-up, cDNA was amplified for 14 cycles using a sample-specific index oligo as a primer. A final round of double-sided size selection using SPRIselect beads followed. Final library size and quantity was determined using an Agilent Tapestation High Sensitivity D5000 (Agilent, USA) and a Qubit dsDNA High Sensitivity Assay kit (Thermofisher, USA), respectively. Additional library quantification was done using the Kapa Library Quantification kit for Illumina Libraries (Roche, USA).