title: GSM4367194: DPPA1: adult Debao pony pituitary1; Equus caballus; RNA-Seq
library_construction_protocol: Each sample was individually ground (mortar and pestle, under continuous liquid N2 chilling) into a fine powder before RNA extraction. Ground samples were stored at -80°C. Total RNA was extracted from 30 mg of ground tissue by using hot phenol method. In brief, cell pellets were resuspended and washed once in Buffer A (50 mM sodium acetate and 10 mM EDTA, pH=5.2). After collecting the cells by centrifugation, the pellets were resuspended in Buffer A containing 1% SDS and immediately added to hot phenol. After incubation at 65°C for 5 minutes followed by centrifugation for 10 minutes at 4°C, the RNA-containing supernatants were transferred to a new tube for ethanol precipitation, washed and then dissolved in DE PC-treated water. The RNA was further purified with two phenol-chloroform treatments and then treated with RQ1 DNase (Promega) to remove DNA. The quality and quantity of the purified RNA we redetermined by measuring the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus (BioRad). The integrity of the RNA was further verified by 1.5% agarose gel electrophoresis. Ribosomal RNAs were removed from the RNA samples (10 μg) using a RiboMinus rRNA depletion kit (Ambion), and the resulting samples we are used to prepare directional RNA-Seq libraries. The purified mRNAs were then iron -fragmented at 95°C followed by end repair and 5' adaptor ligation. Then, reverse transcription was performed using RT primers containing a 3' adaptor sequence and a randomized hexamer. The cDNAs were purified and amplified, and all 200-500-bp PCR product s were purified, quantified and stored at -80°C until they were used for sequencing. For high-throughput sequencing, libraries were prepared following the manufacturer's instructions, and the Illumina NextSeq 500 was used to collect data from 151 nt single-end sequencing