Perkin elmer labchip gx system

RNA was isolated from tissue followed by a Qiagen RNeasy Micro clean-up procedure (Qiagen, Hilden, Germany) according to the manufacturer's protocol. RNAs were analyzed on Perkin Elmer Labchip GX system (Perkin Elmer, Waltham, MA, USA) for quality assessment with RNA Quality Score ≥ 7.9. cDNA libraries were prepared using 2 ng of total RNA and 1 μl of a 1:50,000 dilution of ERCC RNA Spike in Controls (Ambion® Thermo Fisher Scientific, Waltham, MA, USA) using SMARTSeq v2 protocol [19 (link)] except for the following modifications: 1. Use of 20 μM TSO, 2. Use of 250 pg of cDNA with 1/5 reaction of Illumina Nextera XT kit (Illumina, San Diego, CA, USA). The length distribution of the cDNA libraries was monitored using DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip. All samples were subjected to an indexed PE sequencing run of 2 × 51 cycles on an Illumina HiSeq 2000.
Reads were mapped to MM10 with STAR v2.2.3 and gencode M9 annotation. Gene counts were determined with feature counts, differential gene expression with edgeR in R v 3.1.2 [20 (link)]. Data analysis was performed in pipeline pilot (www.accelrys.com) and plots generated in Spotfire (www.tibco.com).