Ribo zero magnetic gold kit

The mRNA was isolated from total RNA using NEB Next poly (A) mRNA Magnetic Isolation Module (NEB, Ipswich, MA, USA) and rRNA was removed using RiboZero Magnetic Gold Kit (Illumina, CA, USA). RNA library was prepared using KAPA Stranded RNA-SEQ Library Prep Kit (Illumina, CA, USA) according to the instruction manual prior to subject to sequence using Illumina NovaSeq 6000. RNA library was qualified by Agilent 2100 Bioanalyzer and quantified by qPCR analysis. Raw sequencing data were QC qualified and trimmed data were aligned with reference genome/transcriptome (GRCh37). Differentially expressed genes or transcripts were subjected to either pathway or GO analysis. Venn graph and heatmap were generated using R language.

Ribosome profiling and RNA sequencing libraries were prepared using the TruSeq Ribo Profile kit (Illumina, #RPHMR12126) as detailed in the manufacturer’s protocol. Cells for each biological replicate of control, heterozygous, and homozygous cells with or without drug treatment were washed with ice cold PBS and lysed on ice in the presence of 100 μg/mL cycloheximide (Sigma). The cleared lysate was flash frozen and stored at −80 °C until further processing. For ribosomal RNA depletion, the RiboZero magnetic Gold kit (Illumina) was used. Quality of amplified libraries was accessed by capillary electrophoresis with a high sensitivity DNA chip on a 2100 Bioanalyzer (Agilent Technologies) and quantified by quantitative PCR with a sequencing library quantification kit (KAPA Biosystems) on a Roche Light Cycler 480. Multiplexed libraries with 1 % spiked in PhiX control were sequenced on a HiSeq2500 instrument for 50 cycles using version 4 chemistry reagents (Illumina). BCL files were converted to the fastq format for further bioinformatics processing.
The sequencing data from this publication is available at the GEO database.

Both jejunum and mesenteric adipose samples were isolated using RNeasy Plus Mini Kit (Qiagen, Valenica, CA). Quality check was performed using Tapestation RNA HS Assay (Agilent Technologies, Santa Clara, CA) and quantified by Qubit RNA HS assay (ThermoFisher Scientific, Waltham, MA). The RNA integrity number for samples was lower and more variable than expected (2.6 ± 0.6 for intestine and 5.0 ± 1.3 for adipose); thus, ribosomal RNA depletion was performed with Ribo-zero Magnetic Gold Kit (Illumina Inc., San Diego, CA) to ensure RNA isolated was of the highest possible quality. Samples were randomly primed and fragmented based on manufacturer's recommendation (NEBNext® Ultra™ RNA Library Prep Kit for Illumina® Illumina Inc., San Diego, CA). The first strand was synthesized with the Protoscript II Reverse Transcriptase for 40 min at 42°C. Synthesis of second strand cDNA was then carried out and products were amplified by PCR to create the cDNA library for each sample. All remaining steps for library construction were done according to the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (Illumina Inc.). Illumina 8-nt dual-indices were used. Samples were pooled by animal and sequencing was done on a HiSeq with a read length configuration of 150 paired end base pairs with up to 50 million reads per sample.

Total RNA was extracted from leukocytes isolated from peripheral blood. Briefly, approximately 2.5 ml of blood from each subject was static settlement for less than 4 h, followed by centrifugation for 10 min at 3,000×g. The cell pellets were then incubated with 1 ml red blood cell lysate (Solarbio Technology Co., Ltd.) and the resultant lysate was centrifuged for 3 min at 3,000×g. After that, the supernatant was removed and the above steps were repeated three times. Total RNA was extracted using TRIzol, as per the manufacturer’s instructions (Invitrogen). Ribosomal RNA was removed using the Ribo Zero Magnetic Gold kit (MRZG126, Illumina). Quality and integrity of the isolated RNA was verified by NanoDrop (Thermo scientific) and Bioanalyzer (Agilent). OD260/280 ratio ranged between 1.9 and 2.1 and RIN >7.0.

Total RNA was extracted from the leukocytes isolated from peripheral blood. Briefly, approximately 3.5 mL of blood from each subject was incubated at room temperature for less than 4 h, followed by centrifugation for 10 min at 3000 × g. The cell pellet was then incubated with 1 mL red blood cell lysis buffer, and the resultant lysate was centrifuged for 3 min at 3000 × g. Total RNA was isolated from purified leukocyte pellet using the RNeasy kit (Qiagen Inc., Valencia, Calif., #217061). Ribosomal RNA was removed using the Ribo Zero Magnetic Gold kit (MRZG126, Illumina). Quality and integrity of the isolated RNA was verified by NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA) and Bioanalyzer (Agilent Technologies Inc., USA). OD260/280 ratio ranged between 2.0 and 2.2 and RIN > 7.0.

According to the manufacturer’s instructions, we used the Ribo-Zero Magnetic Gold Kit (Illumina, San Diego, CA, USA) and the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) to construct the full transcriptome library. We used the Bioanalyzer 2100 system and qPCR (Kapa Biosystems, Woburn, MA, USA) for quality inspection of the library. We sequenced using the Illumina Hiseq2000/2500 and produced read lengths of 2 × 100/150 bp. The original data was evaluated using fastqc (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/version0.10.1), and compared with the reference genome by the TopHat 2.0 program (http://tophat.cbcb.umd.edu/version2.0.10). After the transcripts of each sample were assembled separately, the transcripts of all samples were summarized and merged using the command of cuffmerge. Then, the ensembl transcript database was used as the annotation reference for mRNA, and the number of sequences in each transcript was standardized according to the total length and samples. The number of sequences in every 1 million pairs to every 1000 bases in exon was used as the expression amount. FDR correction was applied to p value to obtain Q value as follows: The screening criteria for differential mRNA were as follows: P value < 0.05 and Q value ≤ 0.05.