Instrument: Illumina HiSeq 2500
Strategy: miRNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: SINGLE
Construction protocol: Total RNA was isolated with the miRNeasy Mini Kit (Qiagen). 1µg total RNA was electrophoresed to isolate the miRNA fraction from 17 to 29 nct. After extraction from the PAGE gel and precipitation, RNA was resuspended in 4ul Rnase-free water MicroRNAs were ligated with 3' adapters, RT primers annealed, then 5' adapters ligated followed by reverse transcription and amplification including one of 48 different reverse primers. The amplified cDNA constructs were gel purified using QIAEX II Gel Extraction Kit (Qiagen) and eluted in 25µl water. Libraries were quality-checked on the Fragment Analyzer and samples pooled by batches of of 48, 40 and 42 libraries respectively in equal molarity. Each pool was further quantified with Pico-Green. 11pM and used for clustering on the cBot2 (Illumina). Samples were sequenced Single-Read over 51 cycles on HiSeq2500 using the HiSeq Flow Cell v4 (Illumina) and the HiSeq SBS Kit v4 (Illumina). Primary data analysis was performed with the Illumina RTA version 1.18.64 and bcl2fastq-2.16.0.10.