Neoprep

DNA libraries of both SNS DNA (sucrose gradient fractions 3, 4, and 5 + 6 + 7 combined) and genomic DNAs were first sheared by an S2 focused-ultrasonicator (Covaris) for 2 min (Intensity 5, Duty Cycle 10%, Cycles per Burst 200), and then used as inputs to generate sequencing libraries by Ovation Ultralow V1 library prep kits (NuGen). The libraries were subjected to deep sequencing on HiSeq 2000 per manufacturer instructions (Illumina). In two of the three experiments, we used different amplification protocols for library generation with the purpose of estimating possible bias introduced by this crucial but unavoidable step. RNA-seq libraries were made by TruSeq Stranded mRNA library prep kit and NeoPrep (Illumina), and subjected to deep sequencing on HiSeq 2000 per manufacturer instructions (Illumina). Single-end sequenced reads (51 nt) were aligned to the reference Arabidopsis genome (TAIR10), using the Bowtie alignment tool (Langmead et al. 2009 (link)), allowing up to one mismatch and discarding multihit reads. PCR duplicate reads were removed using an in-house script (for full details, see Supplemental Methods).

Hepatic and ileal transcriptome analysis was performed by RNA sequencing on RNA extracts from terminal liver samples, as described[19 (link)]. RNA sequence libraries were prepared with NeoPrep (Illumina, San Diego, CA, United States) using Illumina TruSeq stranded mRNA Library kit for NeoPrep (Illumina, San Diego, CA, United States) and sequenced on the NextSeq 500 (Illumina, San Diego, CA) with NSQ 500 hi-Output KT v2 (75 CYS, Illumina, San Diego, CA, United States). Reads were aligned to the GRCm38 v84 Ensembl Mus musculus genome using STAR v.2.5.2a with default parameters[23 (link)]. Differential gene expression analysis was performed with DEseq215[24 (link)].

To isolate total RNA, TA muscles were placed in 1 mL of RNAsol (Sigma Aldrich) and homogenized using a tissue homogenizer (OMNI International). RNA was extracted after 10 min precipitation in isopropanol and resuspended in RNAse free water and RNase inhibitor (1:20; New England BioLabs, #M0314L). RNA integrity was tested as previously described in Soliman et al.²⁸ using the Agilent Bioanalyzer 2100. RNA libraries were prepared following the standard protocol for the TruSeq Stranded mRNA library kit (Illumina) on the Illumina Neoprep automated microfluidic library prep instrument. Paired end sequencing was performed on the Illumina NextSeq 500 using the High Output 150 cycle Kits.²⁸

Liver transcriptome analysis was performed by RNA sequencing on RNA extracts from terminal liver samples (15 mg fresh tissue), as described in detail elsewhere[22 (link),23 (link)]. The RNA quantity was measured using Qubit^® (Thermo Scientific, Eugene, OR, United States). The RNA quality was determined using a bioanalyzer with RNA 6000 Nano kit (Agilent, Waldbronn, Germany). RNA sequence libraries were prepared with NeoPrep (Illumina, San Diego, CA, United States) using Illumina TruSeq stranded mRNA Library kit for NeoPrep (Illumina, San Diego, CA, United States) and sequenced on the NextSeq 500 (Illumina, San Diego, CA, United States) with NSQ 500 hi-Output KT v2 (75 CYS, Illumina, San Diego, CA, United States). Reads were aligned to the GRCm38 v84 Ensembl Mus musculus genome using STAR v.2.5.2a with default parameters[38 (link)]. Differential gene expression analysis was performed with DEseq237. Genes with a Benjamini and Hochberg adjusted P ≤ 0.05 (5% false discovery rate, FDR) were regarded as statistically significantly regulated. The Reactome pathway database[39 (link)] was used as gene annotation in a gene set analysis using the R package PIANO v.1.18.1[40 (link)], with the Stouffer method and Benjamini-Hochberg adjusted P values (FDR < 0.01).

Hepatic transcriptome analysis was performed by RNA sequencing on RNA extracts from terminal liver samples (15 mg fresh tissue), as described in detail previously[19 (link)]. The RNA quantity was measured using Qubit^® (Thermo Scientific, Eugene, OR, United States). The RNA quality was determined using a bioanalyzer with RNA 6000 Nano kit (Agilent, Waldbronn, Germany). RNA sequence libraries were prepared with NeoPrep (Illumina, San Diego, CA, United States) using Illumina TruSeq stranded mRNA Library kit for NeoPrep (Illumina, San Diego, CA, United States) and sequenced on the NextSeq 500 (Illumina, San Diego, CA, United States) with NSQ 500 hi-Output KT v2 (75 CYS, Illumina, San Diego, CA, United States). Reads were aligned to the GRCm38 v84 Ensembl Mus musculus genome using STAR v.2.5.2a with default parameters[36 (link)]. Differential gene expression analysis was performed with DEseq2[37 (link)]. Genes with a Benjamini and Hochberg adjusted P ≤ 0.05 (5% False Discovery Rate) were regarded as statistically significantly regulated. Enrichment analysis of KEGG pathways were performed using the clusterProfiler package for R[38 (link)].