title: GSM6675026: MEF cells, MIC19 KO, biol rep2 [MIC19-KO-2]; Mus musculus; RNA-Seq
library_construction_protocol: Cells were lysed in 500 μL TRIzol reagent and incubated at room temperature for 5 minutes. 100 μL chloroform was added and fully mixed. Samples were centrifuged at 14,400 rpm for 15 minutes at 4°C and 250 μL aqueous phase containing RNA was transferred to a new tube. 250 μL isopropanol was added to the aqueous phase and mixed well to precipitate RNA. After incubation 10 min at room temperature, RNA pellet was collected through centrifuging at 14,400 rpm for 15 minutes at 4°C and washed with 500 μL 75% ethanol. The RNA pellet was air-dried for 10 min and resuspended in nuclease-free H2O. Mouse tissue samples were first weighted and homogenized as described above with 1ml TRIzol reagent per 100 mg. After homogenization, chloroform was added and the following steps are same with cell samples. A total amount of 2 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using VAHTS mRNA-seq v2 Library Prep Kit for Illumina following manufacturer's recommendations and index codes were added to attribute sequences to each sample. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using fragmentation buffer. First strand cDNA was synthesized and second strand cDNA synthesis was subsequently performed. Remaining overhangs were converted into blunt ends. After adenylation of 3' ends of DNA fragments, adaptor with hairpin loop structure were ligated. Then the PCR was performed. At last, Qubit HS quantification, Agilent 2100 Bioanalyzer/Fragment Analyzer 5300 quality control, the final library size of about 350bp. The libraries were sequenced on an Illumina NovaSeq platform to generate 150 bp paired-end reads, according to the manufacturer's instructions.