title: GSM7669280: 6 weeks female mouse muscle, treated with S/Zn-RS 24h, replication 1; Mus musculus; RNA-Seq
library_name: GSM7669280
library_construction_protocol: 1. put the tissue block directly into the mortar, add a small amount of liquid nitrogen, grind quickly, wait for the tissue to soften, add a small amount of liquid nitrogen, and then grind until the grinding is complete. Then, add the tissue sample to 1 ml of Trizol at 50-100 mg and transfer it to a centrifuge tube.2. Add chloroform, which accounts for 1/5 of the total volume. Cap the centrifuge tube tightly and shake vigorously for 15 sec and let stand at room temperature for 2-3min. Then centrifuge at 12,000 rpm at 4°C for 10-15 min. Carefully pipette the upper aqueous phase into a new centrifuge tube and add equal volumes of isopropyl alcohol. Mix upside down and leave at room temperature for 10min. Then centrifuge at 12,000 rpm at 4°C for 10 min. Carefully discard the supernatant and add 1 ml of 75% ethanol prepared with DEPC water. Wash the tube cap and wall well and flick the tube bottom, let the pellet float. Centrifuge at 12,000 rpm at 4°C for 3 min, discard supernatant. Leave at room temperature for 2-3min and let dry. Add an appropriate amount of RNase free water (DEPC water), after complete dissolution, take a small amount for testing, and store the rest at -80°C. 3. RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) . RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA, Catalog #: E7530L) following manufacturer's recommendations and index codes were added to attribute sequences to each sample.Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H.Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEB Next Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beverly, USA). Then 3 µL USER Enzyme (NEB, USA) was used with size selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent 5400 system（Agilent， USA） and quantified by QPCR (1.5 nM). The qualified libraries were pooled and sequenced on Illumina platforms with PE150 strategy in Novogene Bioinformatics Technology Co., Ltd (Beijing, China), according to effective library concentration and data amount required.