GSM9110791: INTRUST_07, timepoint: C2D1; Homo sapiens; RNA-Seq
Samples werethen homogenized using a Retsch MM400 bead mill (4 min, 30 Hz). Tissue homogenateswere centrifuged at 4°C for 5 min at 6000 rpm, and 200 µL of the supernatant wassubsequently used for RNA extraction with the Maxwell RSC 16 (Promega). Finally, RNAwas subjected to quantity and quality control using Qubit HS RNA Assay Kit (Thermo FisherScientific) and 4200 Tapestation with High Sensitivity RNA ScreenTape (AgilentTechnologies). Library preparation was performed using the Illumina Stranded mRNA Prep Ligation kit(Illumina) following the manufacturer's protocol. All sequencing libraries were constructedfrom 100 ng of total RNA according to the manufacturer&#39;s instructions. Briefly, the protocolselects and purifies poly(A) containing RNA molecules using magnetic beads coated withpoly(T) oligos. Poly(A)-RNAs are fragmented and reverse transcribed into the first cDNAstrand using random primers. The second cDNA strand is synthesized in the presence ofdUTP to ensure strand specificity. Resulting cDNA fragments are purified with AMPure XPbeads (Beckman Coulter), adenylated at 3′ ends, and then ligated with uniquely indexedsequencing adapters. Ligated fragments are purified and PCR amplified to obtain the finallibraries. The quality and quantity of the libraries were verified using the Qubit dsDNA HSAssay Kit (Thermo Fisher Scientific) and a 4200 TapeStation with High Sensitivity D1000ScreenTape (Agilent Technologies). Libraries were then sequenced using a NextSeq2000sequencer (Illumina). 50 million pair-end reads (100 bp; Rd1:51; Rd2:51) were sequencedfor each sample and demultiplexed using bcl2fastq. RNA-seq sequencing was carried out atthe Genomics Unit of the Center for Applied Medical Research (CIMA, Universidad deNavarra).