GSM4745104: Naive_day0_1_S1; Mus musculus; RNA-Seq
Mouse T cells were resuspended in TrizolTM Reagent (Thermo Fisher Scientific). Total RNA were isolated per manufacturer's instruction and resuspended in RNase free water. RNA was quantified with Qubit® RNA HS Assay Kit (Thermo Fisher Scientific) and RNA intergrity was verified with Agilent RNA 6000 Nano kit on the 2100 Bioanalyzer (Agilent Technologies) according to the manufacturers' protocol. RNA-Seq libraries were prepared from 65 ng of total RNA with ERCC ExFold RNA Spike-In Mix 1 (Life Technologies) of 1 μL of 1:5000 (v/v) dilution with TruSeq® Stranded mRNA Library Prep for NeoPrepTM kit as per the NeoPrep Library Preparation System Guide on the instrument (Illumina). The libraries were diluted 1:5 (v/v), with 1 μL used to check the expected size (~300 bp) and the samples' purity were verified with the Agilent High Sensitivity DNA Kit as manufacturer's recommendation (Agilent Technologies). One μL of the diluted libraries were used to quantify as recommended in the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) with standard range from 0 to 14 ng/ well. Pooled libraries of 1 nM were denatured and diluted as per the Standard Normalization Method for the NextSeq® System (Illumina). Final loading concentration of 1.7 pM and 1% (v/v) PhiX was used with the NextSeq® 500/550 High Output Kit v2 (150 cycles) (Illumina Samples were sequenced paired-end with 75 cycles of each read  and single index of 6 cycles.