GSM6126088: CD4 T Cells cultured with IL-12+IL-21, GFPneg rep3; Mus musculus; RNA-Seq
Samples were collected into ~200ul of lysis buffer (TCL+BME) and stored in 1.5mL Eppendorf tubes. In preparation for library construction, samples were thawed briefly at RT and ~20ul of each was transferred to a 96-well plate. The construction of the full-length RNA-seq libraries followed a modified SMART-Seq2 protocol (Picelli et al., 2013). After first strand cDNA synthesis and amplification, the cDNA was purified with a 0.7x single sided SPRI.  Each sample was normalized to 0.20ng/ul and then tagmented and indexed using the Nextera XT DNA library prep kit (Illumina). The individually barcoded libraries were then pooled and a final quality control was done on the BioAnalyzer (Agilent). The pool was normalized to 4nM, denatured using 0.2 N NaOh and 0.2 M Tris-HCL and diluted in HCL to prepare for sequencing. 1300ul of the final diluted pooled library was loaded for sequencing on a NextSeq 500/550. Paired end sequencing was performed using an Illumina 75 cycle Nextseq 500 high output V2 kit in accordance with Illumina protocol (Reads 38 cycles x2, Index 8 cycles x2).