title: GSM9135511: LCMV Cl 13 Infected WT mice; Mus musculus; RNA-Seq
library_construction_protocol: Bone marrows were collected from uninfected WT and LCMV Cl 13-infected WT and Sphk2–/– mice at 3 dpi. Bone marrow cells were isolated as mentioned above with little modification in the isolation and suspension buffer. Cells were passed through a 40 µM cell strainer twice to remove debris and aggregates. Total bone marrow cell suspensions were resuspended in PBS+0.04% BSA buffer. Finally, 1×106 cells/ml with >85% viable bone marrow cell suspensions were prepared and immediately handed over to the university DNA core facility for scRNA-seq. Libraries were constructed by following the manufacturer's protocol with reagents supplied in 10× Genomics Chromium Next GEM Single Cell 3′ Kit v3.1.  Briefly, cell suspension concentration and viability were measured with a Cellometer K2 (Revvity) stained with an acridine orange/propidium iodine dye mix (Invitrogen).  Subsequently, cell suspension (targeting 10,000 cells) combined with reverse transcription master mix was loaded on a Chromium Next GEM chip G along with gel beads and partitioning oil to generate gel emulsions (GEMs).  GEMs were then transferred to a PCR strip tube, followed by the reverse transcription, which was performed on a Veriti thermal cycler (Applied Biosystems) at 53°C for 45 minutes.  cDNA was amplified for 11 cycles and purified using AxyPrep MagPCR Clean-up beads (Axygen).  cDNA fragmentation, end-repair, A-tailing and ligation of sequencing adaptors were performed according to the manufacture's specifications.  Library concentration was measured with a Qubit HS DNA kit (Invitrogen) and fragment size was measured on a 5200 Fragment Analyzer (Agilent).  Libraries were pooled and sequenced on a NovaSeq X (Illumina) to generate 50,000 reads per cell with a sequencing configuration of 28 base pair (bp) on read 1 and 100 bp on read 2.