title: GSM8740239: Ac4ManNAz-treated, rep 1; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated according to manufacturer instruction of the Aurum™ Total RNA Mini Kit (Bio-Rad, Cat# 7326820). Afterwards, RNA concentration was measured using a Nanodrop™ 2000 spectrophotometer (Thermo Scientific™), where samples with ratio of absorbance at 260/280 nm >1.8 and concentration >100 ng/µl (in 30 µl) were further used for bulk RNA sequencing Total RNA is used as input material for the RNA sample preparations. rRNA is removed from total RNA by using specific probes. Fragmentation is carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer. First strand cDNA is synthesized using random hexamer primer and reverse transcriptase. Then the second strand of cDNA is synthesized by adding buffer and dNTPs (dTTP in dNTP is replaced by dUTP). The synthesized double-stranded cDNA is treated by terminal repair, adding A, connecting adaptors, and PCR enrichment is performed after fragment selection. In order to select cDNA fragments with a length of 370-420bp, PCR products are purified with AMPure XP beads to obtain strandspecific library. After the library construction is completed, preliminary quantification is carried out using Qubit. Subsequently, the inserted fragments of the library are detected. Once the inserted fragments meet expectations, the effective concentration of the library is accurately quantified using qRT-PCR to ensure the quality of the library. The qualified libraries are pooled and sequenced on Illumina platforms, according to effective library concentration and data amount required.
identifiers.namespace: GEO
identifiers.id: GSM8740239_r1