title: GSM7874682: pMacs, R1441C KI, M, O, untreated, replicate 3; Mus musculus; RNA-Seq
library_name: GSM7874682
library_construction_protocol: RNA from approximately 2-4 x 106 cells was isolated. RNeasy mini kit (Qiagen) was used according to manufacturer's instructions. Briefly, 10 µL of β-mercaptoethanol was added to every 1 mL of RLT buffer. 350 µL was added to each well and cells homogenized manually with a mini cell scraper. Cell lysate was transferred to an RNAase free Eppendorf and an equal volume of 70% ethanol was added to each sample. Samples were loaded into supplier columns and centrifuged at 11,000 x g for 30 seconds and flow through discarded. 350 µL RW1 buffer was added to each column and centrifuged at 11,000 x g for 15 seconds and flow through discarded. RW1 buffer was added and columns centrifuged at 11,000 x g for 15 seconds. RPE buffer was added to columns, centrifuged for 11,000 x g for 30 seconds, repeated, and 30 µL RNase-free water was added and RNA was eluted. RNA concentration was quantified and 260/230 and 260/280 ratios recorded using a spectrophotometer. RIN values were assessed to ensure RNA integrity using Agilent RNA 6000 Nano Kit (Agilent) and Agilent 2100 Bioanalyzer RNA was sent to the Interdisciplinary Center for Biotechnology Research (ICBR) core at the University of Florida, where library preparation was performed following the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina manual and 2x150bp paired-end sequencing completed on an Illumina NovaSeq 6000 (Illumina).