Sequencing of miRNAs of tuberal hypothalamus (arcuate nucleus and median eminence) of single male wistar rat (28 days) born to dam fed high-fat diet
The goal of this experiment was to characterize miRNA populations of tuberal hypothalamus of single male wistar rats of 28 days. This experiment is one of the seven biological replicates sequenced. Small (<150-200 bases) RNAs were isolated from Qiazol homogenates (Qiagen) using the RNeasy mini kit (Qiagen). RNAs were size-fractionated on a 17% Tris-Borate-EDTA urea (8M) polyacrylamide  gel. Small RNAs (18-36nt) were recovered from gel slices and used to  build a cDNA library following an Illumina-like protocol. Small RNAs were ligated to adenylated 3'adaptor (CCCTTCTCGTATGCCGTCTTCTGCTTG). Following ligation to 5'adaptor (rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC) and reverse transcription with RT-primer (CAAGCAGAAGACGGCATACGA), amplification reaction was performed using complementary oligonucleotides to the adaptors but with the extra dinucleotide (CA) at position 7 of the 3' adaptor to tag the library (CAAGCAGAAGACGGCATACGTGAGAAGGG). 16 PCR cycles at 98°C 20s, 55°C 30s and 72°C 25s. PCR products were size-fractionated on a 6% Tris-Borate-EDTA polyacrylamide gel to purify the small RNAs-derived cDNA of 90-95bp from the adaptor-dimer byproduct (74bp). Sequencing kit:36 cycles Sequencing kit v5 (FC-104, 5001, Illumina).
Universite Paris sud
TH-HF28-7