RNA-sequencing (RNA-seq) was performed by Omega Bioservices. Bulk RNA of 9 tissues (heart, hippocampus, hypothalamus, kidney, liver, prefrontal cortex, skeletal muscle, small intestine, and spleen) (4 samples per group) was isolated with the QIAGEN miRNeasy mini extraction kit (QIAGEN) and cDNA was synthesized with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RNA quality was assessed with a Bioanalyzer 2100 (Agilent Technologies). Poly(A)-selected RNA-seq libraries were generated using the Illumina TruSeq RNA Sample preparation kit v2. RNA-seq was performed in a 150-bp paired-end format with a minimum of 40 million reads per sample on the Illumina HiSeq platform according to the manufacturer’s instructions. RNA-seq reads were filtered and trimmed for quality scores >30 using a custom python script. The filtered reads were aligned to Mus musculus GRCm38 using RSEM (v1.2.12) (58) with Bowtie2 (v2.2.0) (59) (command: rsem-calculate-expression -p 12 –phred33-quals –seed-length 25 –forward-prob 0 –time –output-genome-bam – bowtie2). RSEM calculates expected counts and transcript per million (TPM). The expected counts from RSEM were used in the Bioconductor edgeR 3.20.9 package (60) to determine differentially expressed genes.
Analysis performed by Ann Wells
The Carter Lab The Jackson Laboratory 2023
ann.wells@jax.org