TruSeq RNA Library Prep Kit v2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	0.1 - 1 ug total RNA or 10 - 400 ng previously isolated mRNA (from species with polyA tails)

Protocol tips
0.1 - 1 ug total RNA or 10 - 400 ng previously isolated mRNA (from species with polyA tails)

Publication protocol

RNA quality was assessed by using Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer according to the manufacturer’s instructions. Qualified total RNA (RIN > 9, 200 ng) from each sample was processed by following TruSeq RNA Library Prep Kit v2 protocol (Illumina, San Diego, CA). In brief, poly-A containing mRNA purified from each total RNA samples was applied to cDNA library construction. The libraries were sequenced at pair end with read length of 100 bp on Illumina HiSeq 4000 platform at a depth of more than 40 million reads. The experiments were carried out by BGI Americas (Cambridge, MA), a fee-for-service provider.

ArrayStudio version 8.0 (Omicsoft, Cary, NC) was applied to quality control (QC) raw RNA sequencing reads, map reads to genome, quantify gene expression, and test expression changes. In brief, low-quality bases and adaptors were trimmed and reads less than 25 bases were discarded. Remaining reads were mapped to mouse GRCm38 genomes (https://www.ncbi.nlm.nih.gov/grc/mouse) using Omicsoft sequence aligner (OSA) [18] of the ArrayStudio software. Gene expression read count and TPM (Transcript Per kilobase Million) were calculated based on mouse version m10 of GenCode gene models (https://www.gencodegenes.org/mouse_releases/10.html/). Samples in each group were QCed-based overall gene expression consistency, and outliers were removed before downstream analysis. Robust center scale was applied to normalize data in all heat maps.
We deposited raw read fastq and sample metadata files in NCBI with BioProject ID PRJNA407656.

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