For each condition, 200 cells were collected at 4 ˚C in 8 μL of lysis buffer composed by 0.2% Triton X-100, 2 U/ul of recombinant RNase inhibitor (Clontech/Takara), 5 mM dNTP mix (Life Technologies) in 0.2 ml PCR tube (Maxymum recovery, Axygen). Right after sort, tubes were vortexed at medium speed, spun for 5 minutes at >2000 g and stored at -80˚C until the completion of the whole set of samples. Purified total RNA (≈5 ng) was amplified following the Smart-seq2 protocol (Picelli et al., 2014; Rosales et al., 2018). Briefly, mRNA was captured using poly-dT oligos and directly reverse-transcribed into full-length cDNA using the described template-switching oligo (Picelli et al., 2014; Rosales et al., 2018). cDNA was amplified by PCR for 18 cycles and purified using AMPure XP magnetic bead (0.9:1 (vol:vol) ratio, Beckman Coulter). For each sample, 1 ng of cDNA was used to prepare a standard NextEra XT sequencing library (NextEra XT DNA library prep kit and index kits; Illumina). Barcoded Illumina sequencing libraries (Nextera; Illumina) were generated utilizing an automated platform (Biomek FXP, Beckman Coulter). Both whole-transcriptome amplification and sequencing library preparations were performed in a 96-well format to reduce assay-to-assay variability. Quality control steps were included to determine total RNA quality and quantity, the optimal number of PCR preamplification cycles, and fragment library size. Samples that failed quality controls were eliminated from downstream steps. Library that passed strict quality controls were pooled at equimolar concentration, loaded and sequenced on the Illumina Sequencing platform, HiSeq2500 (Illumina). Libraries were sequenced to obtain more than 8 million 50-bp single-end reads (HiSeq Rapid Run Cluster and SBS Kit V2; Illumina) mapping uniquely to mRNA reference, generating a total of about 204.3 million mapped reads (median of about 8.6 million filtered mapped reads per sample). RNA-Seq (Microscaled (SmartSeq2) cDNA amplification; HISeq 2500 illumina, Single-end 50bp)
GEO Accession
GSM3265207