GSM5356096: pool4_cd45; Mus musculus; RNA-Seq
Two days after the anti-CD40 treatment time point, mice were sacrificed and livers and tumors were isolated for generating single cell suspensions. Tissues were enzymatically digested, passed through a cell strainer, and cells were stained for CD45 and labeled with 7AAD; CD45+ cells or IL-12-EYFP+ cells were sorted using a BD FACSAria sorter. InDrops single cell RNA sequencing was performed. A microfluidic device was used to co-encapsulate individual cells and polyacrylamide beads carrying barcoding reverse transcription (RT) primers and lysis reagents into 2-3 nl droplets, followed by primer release and RT at 50°C. After the RT reaction, droplets were broken, and the resulting barcoded cDNA was taken through the following sequencing library preparation steps 1) second strand synthesis, 2) in vitro transcription providing linear amplification of the material, 3) fragmentation of the amplified RNA, 4) a second reverse transcription using random hexamer primers bearing a universal PCR primer annealing site, and 5) indexing PCR, yielding a sequencing-ready library.