GSM8912418: RNA AD,disease,rep3; Homo sapiens; RNA-Seq
Total RNA was isolated and purified from 13 tissue samples using TRIzol (Invitrogen, CA, USA). Next, the quantity and integrity of the total RNA were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and a Bioanalyzer 2100 system (Agilent, CA, USA), respectively. Samples with concentrations > 50 ng/µL, RIN values > 7.0, OD 260/280 > 1.8, and total RNA > 1 µg were considered suitable for downstream experiments. Poly(A) RNA was purified from 1 µg of total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) through two rounds of purification. Subsequently, the poly(A) RNA was fragmented into small pieces using the Magnesium RNA Fragmentation Module (NEB, cat. e6150, USA) at 94°C for 5-7 minutes. The cleaved RNA fragments were then reverse-transcribed to create cDNA using SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA). What's more, Polymerase Chain Reaction (PCR) amplification was performed under the following conditions: initial denaturation at 95°C for 3 minutes; 8 cycles of denaturation at 98°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 30 seconds; followed by a final extension at 72°C for 5 minutes. The average insert size for the final cDNA library was 300±50 bp. Sequencing was carried out on the Illumina NovaSeq 6000 platform using the paired-end 150 bp (PE150) sequencing mode.