GSM7749722: D728,48h,rep4; Homo sapiens; RNA-Seq
Total RNAs were extracted from the tumor xenografts using the TRIzolTM Reagent (ThermoFisher Scientific). RNA libraries were prepared for sequencing using KAPA mRNA HyperPrep Kit Illumina® Platforms protocol A total amount of 1 μg total RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using KAPA mRNA HyperPrep Kit (KAPA Biosystems, Roche, Basel, Switzerland) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using magnetic oligo-dT beads. Captured mRNA was fragmented by incubating at a high temperature in the presence of magnesium in KAPA Fragment, Prime and Elute Buffer (1x). First strand cDNA was synthesized using random hexamer priming. Combined 2nd strand synthesis and A-tailing, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), incorporated dUTP into the second cDNA strand, and added dAMP to the 3' ends of the resulting dscDNA. dsDNA adapter with 3'dTMP overhangs were ligated to library insert fragments to generate the library fragments carrying the adapters. In order to select cDNA fragments of preferentially 300~400bp in length, the library fragments were purified with KAPA Pure Beads system (KAPA Biosystems, Roche, Basel, Switzerland). The library carrying appropriate adapter sequences at both ends was amplified using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Roche, Basel, Switzerland) along with library amplification primers. The strand marked with dUTP in not amplified, allowing strand-specific sequencing. At last, PCR products were purified using KAPA Pure Beads system and the library quality was assessed on the Qsep 100 DNA/RNA Analyzer (BiOptic Inc., Taiwan). The library quality was assessed on the Qubit® 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina NovaSeq6000 platform and 150 bp paired-end reads were generated.