title: GSM7913190: Blood_AD_20; Homo sapiens; RNA-Seq
library_construction_protocol: The blood was collected in PAXgene Blood RNA tubes. Total RNA was extracted following the protocol developed by this lab to isolate RNAs of different sizes and low concentration (Paz S, Mauer C, Ritchie A, Robishaw JD, Caputi M. A simplified SARS-CoV-2 detection protocol for research laboratories. PLoS One. 2020 Dec 18;15(12):e0244271). RNA sample quality and quantity were determined utilizing an Agilent TapeStation system and a NanoDrop spectrophotometer. The samples utilized for the cDNA library preparations had an RNA integrity number (RIN) ≥ 7 and an RNA concentration ≥ 30 ng/µL. Ribosomal RNA was removed by Epicentre TruSeq Stranded Total RNA Library Prep Gold/illumina/20020599 A total amount of 2 μg RNA per sample was used as input material for the RNA sample preparations. Firstly, ribosomal RNA was removed by Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA), and rRNA free residue was cleaned up by ethanol precipitation. Subsequently, sequencing libraries were generated using the rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations. Briefly, fragmentation was carried out using divalent cations under elevated temperature in first strand synthesis reaction buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37° C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.