GSM3895498: Nuclear RNA-seq 1R Rep #2; Arabidopsis thaliana; RNA-Seq
Nuclei were purified by INTACT and RNA was extracted by use of Qiagen Rneasy micro RNA-seq library preparation for nRNA, polyA, and TRAP RNA was performed as described in Kumar et al., 2012.  In brief, RNA was fragmented and primed for first strand synthesis with random hexamers (Invitrogen).  First strand synthesis was performed, followed by second strand synthesis, end repair, and A-tailing. Universal sequencing adapters were ligated to cDNA molecules.  An initial enrichment and adapter extension PCR was first performed on 0.25x volume of the ligated products for each sample to determine amplification cycle number to prevent overamplification of sequencing libraries.  A final enrichment and adapter extension PCR was performed with 0.25x starting volume for the specified cycle number (13-15 cycles) for each sample. Following enrichment, purification, size selection of libraries, and sequencing was performed as described for ChIP-seq.