GSM4861243: N0972: BALBc Liver digested  NKT1 CD4+ 23.000 cells; Mus musculus; RNA-Seq
CD4i+iNKT1 cells were lysed immediately following flow cytometry sorts, RNA was prepared using RNeasy Plus Mini Kit (QIAGEN) 1-5ng of total RNA were used for library preparation with the 'SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian' (#634413; Takara/Clontech) according to conditions recommended in user manual #063017. Generated libraries were barcoded by dual indexing approach and were finally amplified by 11 cycles of pcr. Fragment length distribution of generated libraries was monitored using 'Bioanalyzer High Sensitivity DNA Assay' (5067-4626; Agilent Technologies). Quantification of libraries was performed by use of the 'Qubit® dsDNA HS Assay Kit' (Q32854; ThermoFisher Scientific).  Equal molar amounts of twelve to thirteen individually barcoded libraries were pooled for one sequencing run. Accordingly, each analyzed library constitutes 8.3% (7.7%) of overall flowcell capacity. The library pool was denatured with NaOH and was finally diluted to 1.5pM according to the Denature and Dilute Libraries Guide (Document # 15048776 v02; Illumina). 1.3 ml of denatured pool was loaded on an Illumina NextSeq 550 sequencer using a High Output Flowcell for 75bp single reads (#FC-404-2005; Illumina).