title: Testis-2
design: RNA library for lncRNA-seq was prepared as rRNA depletion and stranded method. Briefly, the ribosomalRNA was depleted from total RNA using the rRNA Removal Kit following manufacturers instruction. RNAwas then fragmented into 250~300 bp fragments, and first strand cDNA was reverse-transcribed usingfragmented RNA and dNTPs (dATP, dTTP, dCTP and dGTP). RNA was degraded using RNase H, andsecond strand cDNA was synthesised using DNA polymerase I and dNTPs (dATP, dUTP, dCTP and dGTP). Remaining overhangs of double-strand cDNA were converted into blunt ends via exonuclease/ polymeraseactivities. After adenylation of 3 ends of DNA fragments, sequencing adaptors were ligated to the cDNA.In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purifiedwith AMPure XP system. Uridine digestion was performed using Uracil-N-Glycosylase, which was followedby the cDNA amplification using PCR. After library construction, the concentration of library was measured by the Qubit fluorometer andadjusted to 1ng/uL. Agilent 2100 Bioanalyzer was deployed to examine the insert size of the acquiredlibrary. At last, the accurate concentration of cDNA library was again examined using qPCR. Once theinsert size and concentration of the library was identical, the samples can then be subjected for sequencing.