title: GSM6723608: ZN_D1_1; Cricetulus griseus; RNA-Seq
library_construction_protocol: Extractions of RNA were carried out using the Qiagen RNeasy Plus Mini Kit (Qiagen, Cat. No. 74136). Extractions were performed according to the manufacturer's protocol for “Purification of Total RNA from Animal Cells” with the following changes: samples were prepared per treatment protocol above, option 3c was followed for Step 3, molecular grade ethanol was used for Step 5, optional Step 10 was used, and Dnase/Rnase-free water was heated to 37 ˚C and allowed to incubate on the column for 5 min for elution. Quantification of RNA was performed by measuring absorbance at 260 and 280 nm using a nanospectrophotometer.  Multiple aliquots of 100 ng/µL were prepared and stored at -80 ˚C until ready for analysis. Concentrations of RNA and RNA Integrity Numbers (RIN) were determined by Qubit fluorometer (Invitrogen) and 2100 Bioanalyzer (Agilent), respectively. After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. For prokaryotic organisms or eukaryotic organisms' long-non-coding libraries,  rRNA is removed using the Ribo-Zero kit that leaves the mRNA. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using  mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the  second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and  PCR enrichment. The quality control of library consists of three steps: (1) Qubit 2.0: tests the library concentration preliminarily. (2) Agilent 2100: tests the insert size. (3) Q-PCR: quantifies the library effective concentration precisely.