title: BGISEQ-500 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℃. Firstly, we used the PAGE electrophoretic glue cutting to separate the total RNA into 18-30nt RNA. Then use 5-adenylated, 3-blocked single DNA binding to bind the above 18-30nt RNA to the 3' joint of the RNA. Secondly, we added RT primer to hybrid with the 3'joint binding to RNA and to hybrid with the redundant 3'joint. The 5'joint was binded to 5'joint of products afterwards. Because the connectors were preferentially attached to single stranded molecules, RT primers were used for reverse transcription extension to synthesize a strand of cDNA. We amplified cDNA with high sensitivity polymerase, enriched cDNA with 3 'and 5' connectors at the same time, and amplified library yield. PCR products in the range of 100-120bp were isolated by PAGE electrophoresis, and byproducts such as primer dimer were removed