GSM2345211: e10-5-L26-9-dorsal; Mus musculus; RNA-Seq
Immediately following Laser micro-dissection, the collection tubes were capped and vortexed.  After addition of 300 μl RLT buffer to each tissue sample (total 350 μl), the RNA was prepared using the RNeasy Micro Kit (Qiagen #74004) as per instructions.  An on column DNase digestion was included in RNA preparation. Total RNA was processed for library construction by Cofactor Genomics (St. Louis, MO) according to the following procedure: 700-1000pg of RNA was amplified using the Ovation RNA-Seq System v2 system (NuGEN, San Carlos, CA). Double-stranded cDNA was sheared to the desired size (175-200bp) using the Covaris S2 (Covaris, Woburn, MA). Indexed adaptors (Illumina truseq barcodes 1-8 ) were ligated to 500ng cDNA.  Ligation reactions were incubated at 20oC for 15 minutes, and the adaptor-ligated DNA was then size-selected (175-200bp) on a 2% SizeSelect™ E-Gel (Invitrogen, Carlsbad, CA) and amplified by PCR. PCR cycling conditions used were: 45 seconds at 98oC; (15 seconds @ 98oC, anneal for 30 seconds at 60oC, extend for 30 seconds at 72oC) – repeat 7X; 60 seconds at 72oC.  Library quality, quantity and insert size range was assessed with an Agilent Bioanalyzer.