title: HiSeq X Ten paired end sequencing; Pnldc1 Total RNA-seq of P18 Testis
design: Pnldc1 Total RNA-seq of P18 Testis
library_construction_protocol: Euthanize mice using carbon dioxide (CO2) for 1-2 min or until breathing stops. Next, perform cervical dislocation on each mouse.Extract the mouse testis tissue and remove the capsule Total RNA was isolated using RNeasy mini kit (Qiagen, Germany). Strand-specific libraries were prepared using the TruSeq® Stranded Total RNA Sample Preparation kit (Illumina, USA) following the manufacturer's instructions. Briefly, ribosomal RNA was removed from total RNA using Ribo-Zero rRNA removal beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under 94℃ for 8 min. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library. Purified libraries were quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated byAgilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration.