title: GSM2427926: 3_7d miRNA-Seq; Homo sapiens; miRNA-Seq
library_construction_protocol: miRNA-Seq: 1 microgram total RNA of every sample was processed following the Illumina TruSeq small RNAseq library prep protocol (cat. RS-200- 0012 – 0024). A single stranded 3’-RNA adapter and a single stranded 5’-RNA adapter were ligated to the total RNA of all samples. After reverse transcription, PCR-amplification of the resulting cDNA was performed using a unique primer pair for each sample. Each library was quantified before gel-purification based on Kapa® quantification (Kapa Biosystems®, cat KK4835) using 3 independent 10 -6 dilutions. The concentrations ranged 1.3-2.9 microgram cDNA with an average of 2.1 microgram. The fragment size distribution of each library was quality-controlled using the Agilent Tapestation® 2200. The 20 libraries were pooled and electrophoresed on a 10% acrylamide gel for purification of fragments spanning 140-160 base pairs. This fragment size covers the adaptor-ligated mature miRNAs, where the adaptors account for 120 bp. The content of this extract was quality-controlled using the Agilent Tapestation® 2200. The average library size was 150 bp. The concentration was determined with the Kapa® library quantification kit (Kapa Biosystems®, cat KK4835) using 3 independent 10 -6 dilutions of the gel-purified library pool. The concentration was normalized to 2nM, spiked in with 10% PhiX library and loaded onto two flow cell lanes of a rapid HiSeq2500 run at a concentration of 13 pM. Each template molecule was clonally amplified using a Cbot2® system (Illumina). Sequencing by synthesis was performed on the Illumina HiSeq2500®, using a strategy of 50bp single-end. The total read depth ranged 4.5-13.3 million reads per sample, with an average of 8.4 million reads after subtracting off-target sequences. After cell lysis and total RNA extraction in phenol/chloroform, the aqueous phase was processeded using the RNeasy® Mini Kit (QIAGEN) with DNase digestion as per the manufacturer’s instructions
attributes.tag: GEO Accession
attributes.value: GSM2427926