GSM5234539: Alcoholic hepatitis LDN S28; Homo sapiens; RNA-Seq
40-50ml of whole blood from the AH patients and healthy subjects were collected and diluted with warm RPMI including 1% Anti-anti to 1:1. 36ml of the diluted blood was layered on 12ml of gradient medium Ficoll and centrifuged at 585 xg for 20 minutes. PBMCs were harvested from interphase between plasma and Ficoll and washed with 2% FBS HBSS. After Ficoll was aspirated, red blood cell lysis buffer (BD Bioscience) was added into the RBC/neutrophil fraction. After 15 minutes incubation at room temperature, the tube was centrifuged at 200 xg for 5 minutes and the pellet was washed with PBS and harvested for neutrophils (HDNs). LDNs were isolated from PBMCs using EasySep Human Neutrophil Isolation kit (Stemcell). RNA was extracted from each group of neutrophils through miRNeasy kit (Qiagen). cDNA library was prepared after rRNA removal, using Kapa RNA RiboErase.