GSM7671617: Whole-blood RNA-seq from AD(171104_S01); Homo sapiens; RNA-Seq
RNA was isolated from blood stored in Tempus tubes using the Tempus™ Spin RNA Isolation Kit (reference 4380204, Applied Biosystems). RNA quality was assessed using Agilent RNA 6000 Nano and Pico Chips (Agilent Technologies, Cat.# 5067-1511 and Cat.# 5067-151, respectively) and concentration via Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Cat.# Q32855). Sequencing libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Globin kit (Illumina Inc., Cat.# 20020612) and TruSeq RNA CD Index Plate (Illumina Inc., Cat.# 20019792) following TruSeq Stranded Total RNA Sample Prep-guide (Part # 15031048 Rev. E). Briefly, starting from 500 ng of total RNA, rRNA and globin mRNA were depleted and remaining RNA was purified, fragmented and primed for cDNA synthesis. cDNA first strand was synthesized with SuperScript-II Reverse Transcriptase (Thermo Fisher Scientific, Cat.#18064-014) for 10 min at 25°C, 15 min at 42°C, 15 min at 70°C and pause at 4°C. cDNA second strand was synthesized with Illumina reagents at 16°C for 1 hour. Then, A-tailing and adaptor ligation were performed. Finally, enrichment of libraries was achieved by PCR (30 sec at 98°C; 15 cycles of 10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C; 5min at 72°C and pause at 4°C). Afterwards, libraries were visualized on an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit (Agilent Technologies, Cat. # 5067-4626), quantified using Qubit dsDNA HS DNA Kit (Thermo Fisher Scientific, Cat. # Q32854) and sequenced in a NovaSeq-6000 (Illumina Inc.) by at least 100 million paired-end 100nt reads.