DA330 treated primary human MEPs isolated form bone marrow of normal donor
MEPs were starved for 1.5h in RPMI with 1% BSA followed by incubation in RPMI with 1% BSA and 5 microM DA330 for 2 hours. RNA was isolated with Trizol (Life Technologies) as per the manufacturer’s recommendations and was further facilitated by using linear polyacrylamide (Sigma) as a carrier during the procedure. We treated the total RNA samples with RQ1 RNase free DNase (Promega) to remove minute quantities of genomic DNA if present. DNase treated samples were cleaned up using RNAeasy minelute columns (QIAGEN). 1-10 ng of total RNA was used as input for cDNA preparation and amplification using Ovation RNA-Seq System V2 (NuGEN). Amplified cDNA was sheared using Covaris S2 (Covaris) using the following settings: duty cycle 10%, intensity 5, cycle/burst 100, total time 5 min. The sheared cDNA was cleaned up using Agencourt Ampure XP (Beckman Coulter). 500 ng of sheared cDNA were used as input for library preparation using NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs) as per manufacturer’s recommendations.