GSM6731212: p2080 + doxycyline, replicate 3; Mus musculus; RNA-Seq
Macrophages were homogenized in lysis reagent (RLT buffer, Qiagen, Germantown, MD) using a 20-gauge needle and remaining intact Cn were removed by centrifugation at 750 x g for 5 min at 4 °C. RNA was extracted from the lysates using an RNeasy® Mini Kit (Qiagen), according to the manufacturer's instructions. Genomic DNA was then removed from the total RNA using a Message Clean kit (GenHunter, Nashville, TN) per the manufacturer's instructions. Clean RNA was resuspended in 10 µL diethyl pyrocarbonate (DEPC)-treated water and RNA integrity and quality was appraised using a Qubit 2 fluorometer (ThermoFisher Scientific). The cDNA libraries for RNA sequencing were prepared from 1 µg of isolated RNA using the NEBNext UltraTM Directional RNA Library Prep Kit for Illumina, the NEBNext Multiplex Oligos for Illumina Index Primers, and the NEBNext Poly(A) Magnetic Isolation Module (all from New England BioLabs, Ipswich, MA) in accordance with the manufacturer's instructions.