alias: GSM1919501
title: GSM1919501: AZM microbiome seq_pooled sample; human lung metagenome; OTHER
library_construction_protocol: ion exchange column (Qiagen) Amplicon library preparation was performed using an automated platform (Biomek 4000) using a custom liquid handling method. For each sample, the V4 region of the bacterial 16S rRNA gene was amplified in duplicate reactions, using primer set 515F/806R, which nearly universally amplifies bacterial and archaeal 16S rRNA genes.1,2 Each unique barcoded amplicon was generated in pairs of 25μl reactions with the following reaction conditions: 11μl PCR-grade H2O, 10μl Hot MasterMix (5 Prime Cat# 2200410), 2μl of forward and reversed barcoded primer (5μM) and 2μl template DNA. Reactions were run on a C1000 Touch Thermal Cycler (Bio-Rad) with the following cycling conditions: initial denaturing at 94°C for 3 min followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 58°C for 1 minute, and extension at 72 C for 90 seconds, with a final extension of 10 min at 72°C. Amplicons were quantified using Agilent 2200 TapeStation system and pooled. Purification was then performed using Ampure XT (Beckman Coulter Cat# A63882) as per the manufacturer instructions
identifiers.namespace: GEO
identifiers.id: GSM1919501