GSM8031811: liver,CW1; Hipposideros armiger; RNA-Seq
Commercial RNA simple extraction kit (DP419, TIANGEN, China) was selected to extract the total RNA in liver tissues. 60 mg fresh tissue was ground by high-speed homogenizer (DHFSTPRP-32D, LAWSON, China) with 1 mL lysis buffer RZ to obtain homogenate and then trichloromethane was added. After staying at room temperature for 4 min, the mixture was centrifuged at 12000 rpm to obtain the aqueous phase that was mixed with absolute ethyl alcohol. RD solution was used to remove residual protein impurity in RNA samples and the purified total RNA was stored at RNase-free tubes after repeated centrifugation. When RNA integrity number > 7.0 and A260/280 > 1.8, messenger RNA (mRNA) with poly A was caught using oligo(dT) dynabeads (25-61005, THERMO FISHER, USA) and then broken into fragmentation with the specific kit (E6150S, NEBNEST, USA). Double-strands complementary DNA was synthesized based on the reverse transcriptase and E. coli DNA polymerase I that were provided by LIANCHUAN BIO TECHNOLOGIES Co., LTD (Hangzhou, China). Paired-ends of the complementary DNA were added with a “A” base, which could combine with the adapter sequence. After the second strand was digested, the sequencing library (300±50 bp) was established by amplifying the target sequences.