Sequencing of miRNAs of tuberal hypothalamus (arcuate nucleus and median eminence) of single male wistar rat (4 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 4 days. This experiment is one of the five biological replicates sequenced. Total RNAs were purified using Qiazol lysis reagent (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-HF4-2