GSM1693162: T2DM_ischemic_gastrocnemius_5; Mus musculus; RNA-Seq
Tissues were snap frozen in liquid nitrogen and Total RNA was isolated from both normoxic adductor and ischemic gastrocnemius muscles using RNeasy Fibrous Tissue Mini Kit (QIAGEN Finland, Helsinki, Finland) according to manufacturer's instructions. Total RNA was enriched for Poly (A)+ RNA with MicroPoly (A) Purist Kit (Ambion, Austin, TX, USA). RNA was treated with TURBO DNase (Ambion), fragmented using RNA Fragmentation Reagents (Ambion) and purified by running through P-30 column (Bio-Rad, Hercules, CA, USA). Fragmented RNA was dephosphorylated with Antarctic phosphatase (New England Biolabs, Ipswich, MA, USA) followed by heat-inactivation. Dephosphorylation reactions were mixed with 2 x Novex TBE-Urea sample buffer (Invitrogen), briefly denatured and loaded on a Novex denaturing 15% polyacrylamide TBE-urea gel according to the manufacturer’s instructions. Fragments of 50-300 nucleotides in length were gel purified .  Also the poly (A)+ tailing and cDNA synthesis was performed the next day. However, for reverse transcription oligos with custom barcodes (underlined) were used: 5’phosCA/TG/AC/GTGATCGTCGGACTGTAGAACTCT/idSp/CAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3’. After cDNA synthesis, exonuclease was used to catalyze the removal of excess oligos. Enzyme was inactivated and RNA hydrolyzed by alkaline treatment (100 mM NaOH) and heat (25 min, 95°C). The cDNA fragments of ~150-200 bps were purified on a denaturing Novex 10% polyacrylamide TBE-urea gel (Invitrogen). The recovered cDNA was circularized, linearized, amplified for 8 cycles. The final product was ran on Novex 10%TBE gel, gel purified as above and cleaned-up using ChIP DNA clean & Concentrator Kit (Zymo Research Corporation, Irvine, CA, USA). The library was sequenced for 50 cycles on the Illumina HiSeq 2000 according to the manufacturer’s instructions.
GEO Accession
GSM1693162