GSM9061867: SK_4VIC_F01, placebo, time 0; Homo sapiens; RNA-Seq
Lysate from 1000 cells was processed by the Immunological Genome Project protocol (www.immgen.org): final sort of 1,000 cells directly into 5ɥl lysis buffer (TCL Buffer (Qiagen) with 1% 2-Mercaptoethanol). After sort vortex, keep on ice for 5 minutes, give a quick spin. Freeze on dry ice. Follow Genome Project protocol (www.immgen.org): 5 ul of the lysates are used for each sample for Smart-seq2 library construction, prepared as previously described (Picelli et al., 2013; Picelli et al., 2014) with slight modifications. Briefly, total RNA was captured and purified on RNAClean XP beads (Beckman Coulter). Polyadenylated mRNA was then selected using an anchored oligo(dT) primer (5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3′) and converted to cDNA via reverse transcription. First strand cDNA was subjected to limited PCR amplification followed by Tn5 transposon based fragmentation using the Nextera XT DNA Library Preparation Kit (Illumina). Samples were then PCR amplified for 12 cycles using barcoded primers such that each sample carries a specific combination of eight base Illumina P5 and P7 barcodes and pooled together prior to sequencing. Smart-seq paired-end sequencing is performed on an Illumina NextSeq500 (two full NextSeq runs per batch of 96 samples, for 10M raw reads/sample on average) using 2 x 38bp reads with no further trimming.