title: GSM1174817: Spermatogonia_RNA-seq_rep2; Rattus norvegicus; RNA-Seq
library_construction_protocol: Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA integrity was measured using an Agilent 2100 Bioanalyzer, and only samples with a RIN score >7.0 were further processed. The RNA-seq libraries were prepared using "mRNA-seq sample prep kit" (#RS-100-0801, Illumina Inc.) following Illumina's protocol with some modifications. Briefly, 10 µg of total RNA were hybridized with eukaryote rRNA sequence-specific 5'biotin labeled oligonucleotide probes to selectively deplete abundant ribosomal RNA. The rRNA/5'biotin labeled probe hybrid was removed from the sample with streptavidin-coated magnetic beads ("Ribominus Eukaryote kit for RNA-seq", #A10837-08, Invitrogen). Then, 250 ng of rRNA-depleted RNA were fragmented using divalent cations at 95°C for 5 minutes. The cleaved mRNA fragments were reverse transcribed to cDNA using random primers and SuperScript II reverse transcriptase (# 18064-014, Invitrogen). This was followed by second strand cDNA synthesis using Polymerase I and RNase H. The double stranded cDNA fragments were end-repaired using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments with a Klenow fragment (3' to 5'exo minus). The ends of the DNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were separated by 2% agarose gel electrophoresis and ~200-220bp fragments were excised and purified using QIAquick Gel Extraction Kit (Qiagen) and enriched by PCR (30 sec at 98°C; [10 sec at 98°C, 30 sec at 65°C, 30 sec at 72°C] x 13 cycles; 5 min at 72°C). After PCR amplification, surplus PCR primers were removed by purification using Agencourt AMPure XP beads (#A63881, Beckman). DNA libraries were checked for quality and quantified using 2100 Bioanalyzer (Agilent). Each library was loaded in two lanes of the flowcell at 5pM concentration and clusters were generated using the cluster station and sequenced on the Illumina Genome Analyzer II as paired-end 2x60 base reads. Image analysis and base calling were performed using the Illumina Pipeline version 1.6.