title: Illumina HiSeq 4000 paired end sequencing; RNA-seq of HIV-1 infected long-term non-progressors(LTNPs) and typical progressors(TPs)
design: RNA-seq of HIV-1 infected long-term non-progressors(LTNPs) and typical progressors(TPs)
library_construction_protocol: The study follows the principle of informed consent, and is reviewed and approved by the Ethics Committee of Guangxi Medical University. This research recruit the aimed research people who are meeting the inclusion criteria in Ling shan CDC and Liuzhou CDC.The research people are divided into two groups,named long-term non-progressors（LTNPs）and typical progressors（TPs）in HIV-infected people.Whole peripheral blood mononuclear cells (PBMCs) was obtained from two categories of individuals: long-term non-progressors (LTNPs, n=3) and typical progressors (TPs, n=3).All RNA samples were stored at -80℃ until further processing.  Centrifuge PBMCs with a concentration of 500μL (10 million/mL) into a 1.5mL tube at 1600rpm/min for 6min, and discard the supernatant.After adding 500μL Trizol, the lysed cells were repeatedly blown and placed at room temperature for 20min.Then 100μL chloroform was added,and the mixture was shaken for 15s. The mixture was then allowed to stand for 10min at room temperature.Afterwards，centrifuge the mixture at 14000g,4℃ for 15min.The mixture after centrifugation consists of three layers, the upper aqueous phase, the middle white phase and the lower organic phase.Then, carefully remove the upper water phase into a new 1.5 mL centrifuge tube.Then add 250μL isopropanol and mix it upside-down.Then put the mixture stand at room temperature for 10min.Afterwards,the samples were centrifuged at 14000g,4℃ for 15min.After centrifugation, the observed white precipitation was RNA.Discard the supernatant,then add 500μL 75% ethanol, carefully knock the bottom of the centrifuge tube to separate the precipitation from the bottom,and fully wash the RNA precipitation twice times.Then centrifuge the mixture at 12000g,4℃ for 10min.Discard the supernatant and dry the RNA precipitation in the centrifuge tube in air.Then 30 μL DEPC water was added into the RNA and the RNA was incubated at 65℃ for 10min to dissolve the RNA.Then Agilent 2100 was used to determine RNA concentration, 28S/18S and RIN value.Finally,The extracted RNA was stored at -80℃. The ribosomal rRNA was first removed using a biotin-labeled specific probe (RIbo-Zero ™ rRNA Removal Kit).After purification, RNA is fragmented under certain temperature and ionic conditions.A single strand of cDNA was then synthesized using a random primer and reverse transcriptase from the TruSeq® Stranded Kit,then the double-stranded cDNA was synthesized using DNA polymerase I and RNaseH.During cDNA two-strand synthesis, RNA templates are removed and dTTP is replaced by dUTP.The involvement of dUTP prevents the cDNA double strand from being amplified in the subsequent process because the polymerase is unable to extend across the dUTP site on the template.The double stranded cDNA product was then attached with the \"A\" base and the connector.The ligands are amplified and purified to the final cDNA library.