GSM5591932: B16_Cnt_S1; Mus musculus; RNA-Seq
Reprogrammed cancer cells were sorted through co-expression of eGFP, CD45+ and MHC-II+ at day 3, 5, 7 and 9 post-transductions. LLC co-expressing eGFP and CD45+ were sorted at day 7 and day 9 for mRNA-seq analysis. eGFP transduced cells were stained normally for CD45 and MHC-II and confirmed for null expression of both markers before purification based on eGFP expression. Total RNA from 3000-5000 pre-sorted cells was extracted following TRIzol™ manufacturer's protocol (Invitrogen). cDNA was generated with the SMARTSeq v4 Ultra low input RNA kit (Takara) and amplified with 8 cycles followed by purification by AMPure XP beads. cDNA was analyzed using the Agilent High sensitivity DNA kit on an Agilent Bioanalyzer 2100 (Agilent). Library preparation was performed using the Nextera XT DNA library preparation kit (Illumina) according to the manufacturer's protocol. Following tagmentation of cDNA, forward and reverse indexes were added by 12 cycles of PCR. Normalization was performed by normalizing beads according to manufacturer's protocol and libraries were mixed in equal volumes and sequenced on an Illumina NextSeq 500 (75-bp paired-end).