GSM8565159: MC_D4_TGF_1; Homo sapiens; RNA-Seq
Cells were spun down and resuspended in TCL buffer (Qiagen) with 1% 2-mercaptoethanol. Lysate containing 1,000 cells was transferred into low-bind plates for sequencing through the Broad Institute Genomics Platform SmartSeq2 libraries were prepared according to the SmartSeq2 protocol (Picelli et al, 2013, 2014) with some modifications (Trombetta et al, 2014). Briefly, total RNA was purified using RNA-SPRI beads. Poly(A)+ mRNA was converted to cDNA which was then amplified. cDNA was subject to transposon-based fragmentation that used dual-indexing to barcode each fragment of each converted transcript with a combination of barcodes specific to each sample. Barcoded cDNA fragments were then pooled prior to sequencing. Sequencing was carried out using an Illumina NextSeq500 as paired- end 2x25bp with an additional 8 cycles for each index.