GSM9712662: Ly6C+ GMPs isolated from Idh1-R132H bone marrow, rep 2; Mus musculus; RNA-Seq
Bone marrow was isolated and subjected to Lin-cKit+CD16/32+CD34+Ly6C+ cell sorting. Ly6C+ GMPs were sorted into Arcturus® PicoPure® Extraction Buffer (Applied Biosystems), snap-frozen in liquid nitrogen and stored at -80°C until further processing. Total RNA extraction was performed using the Arcturus® PicoPure® RNA Isolation Kit (Applied Biosystems) according to the manufacturer's protocol. DNA was digested on-column using the RNase-Free DNase Set (Qiagen). RNA was eluted in 10 µl HO and concentrations were determined with a Qubit Fluorometer (Thermo Fisher Scientific) using a Qubit RNA High Sensitivity Kit (Invitrogen). RNA profiles were validated on an Agilent 2100 Bioanalyzer platform (Agilent Technologies) with an Agilent RNA 6000 Pico Kit (Agilent Technologies). RNA concentrations of samples were adjusted to an equal concentration for further processing. cDNA was synthesized in a reverse transcription and template-switching reaction and amplified by pre-amplification PCR. cDNA was purified using Agencourt Ampure XP Beads (Beckaman Coulter) and quality- checked on an Agilent 2100 Bioanalyzer platform (Agilent Technologies) using an Agilent High Sensitivity DNA Kit (Agilent Technologies). Libraries were generated using the Nextera XT DNA Library Prep Kit (Illumina) according to the manufacturer's protocol. cDNA was tagmented and adapter-ligated fragments amplified by PCR. Amplified and indexed libraries were purified using Agencourt Ampure XP Beads (Beckaman Coulter) and quality-checked on an Agilent 2100 Bioanalyzer platform (Agilent Technologies) using an Agilent High Sensitivity DNA Kit (Agilent Technologies). Library concentrations were determined with a Qubit Fluorometer (Thermo Fisher Scientific) using a Qubit DNA High Sensitivity Kit (Invitrogen). For sequencing, libraries were pooled at equimolar ratios.