Sv total rna isolation system protocol

Total RNA of rat INS-1E cells was extracted using the High Pure RNA Isolation Kit (Roche) and cDNA was made using oligo-d(T) and superscript II reverse transcriptase (Invitrogen). Total RNA from murine pancreatic islets and mTECs was extracted using the Single Cell RNA Purification Kit (Norgen) and cDNA synthesized with SuperScript VILO (Invitrogen). Total RNA from whole thymus was extracted using the SV Total RNA Isolation System Protocol (Promega), cDNA was synthesized similarly as cDNA from pancreatic islets and mTECs. qRT-PCR was performed using 4 pmol primers, 0.2 μL cDNA and 5 μL Fast SYBR Green Master Mix (Applied Biosystems) on a StepOnePlus RT-PCR System (Applied Biosystems). The relative fold gene expression was calculated using the delta-delta Ct method. Primers used are summarized in Supplementary Table 1. Normalization was done using the geometric mean of the housekeeping genes (Actin, Rpl27 and Hprt).

To prepare samples for RNA-seq studies, total RNA was isolated from N. triangulifer following the SV Total RNA Isolation System protocol (Promega, WI). RNA quality was assured with Bioanalyzer RNA nano chips (Agilent, CA); Truseq libraries (Illumina, CA) were prepared; and 16 libraries were sequenced with paired-end reads using the Illumina NextSeq 500 at the NCSU GSL.