GSM9100472: MHC-IIlow3; Mus musculus; RNA-Seq
Cells were lysed using Trizol (Invitrogen) and vortexed. For each 1 ml of Trizol, 200 μl of chloroform (Sigma) was added, followed by vortexing. The samples were incubated at room temperature (RT) for 3 minutes and centrifuged at 12,000 rcf for 15 minutes at 4°C. The aqueous phase was collected, and an equal volume of 100% isopropanol was added. After tapping and vortexing, the samples were incubated for 40 minutes at RT. The samples were then centrifuged at 12,000 rcf for 30 minutes at 4°C. The supernatant was removed, and the pellet was washed with cold (-20°C) 75% ethanol (Acros Organics). The samples were centrifuged again at 12,000 rcf for 20 minutes at 4°C. The RNA pellets were dried in a fume hood for up to 10 minutes. The pellets were resuspended in ultrapure DEPC water (Thermo Fisher) and gently vortexed. RNA concentration was determined using the Qubit RNA HS assay (Thermo Fisher), and RNA integrity was assessed using the Agilent RNA 6000 Nano kit (Agilent Technologies). The RiboMinus Eukaryote Kit (Thermo Fisher)  for RNA-Seq was used to deplete ribosomal RNA.The TruSeq Stranded Total RNA kit (Illumina) was used to construct the library for RNA sequencing. The low sample protocol was used and the following adjustments were made to the standard protocol. The first step was cleaning up the RNA with RNAClean XP beads (Beckman Coulter). Then the first strand was synthesized followed by the second strand. CTE for end pair control was not added. The 3' ends were adenylated, the adapter ligated and the DNA fragments were enriched. The library concentration and size was determined with Qubit and Bioanalyzer. The libraries were normalized and pooled.