Kapa stranded rna seq library prep kit

Total RNA was extracted from control and BAMBI-deficient adipose tissues. The transcriptome sequencing experiments were performed by Novogene Company (Beijing, China). The transcriptome library for sequencing was generated using a KAPA-Stranded RNA-Seq Library Prep Kit (Illumina) following the manufacturer’s recommendations. The clustering of the index-coded samples used the KAPA RNA Adapters set1/set2 for Illumina. After clustering, the libraries were sequenced on Illumina HiSeq X Ten platform using a (2 × 150 bp) paired-end module. The differentially expressed genes were identified with p value <0.05 and a fold-change of >1.5 between two groups.

Total RNAs were extracted by TRIzol Reagent (Takara Bio Inc, Dalian, China) following the manufacturer's instructions. The integrity of RNA is tested by agarose gel electrophoresis, while concentrations and quality of total RNA were measured by NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, Delaware). Afterwards, one to two micrograms of total RNA per sample were used for the construction of sequencing library with KAPA Stranded RNA-Seq Library Prep Kit (Illumina). The constructed library was then checked by Agilent 2100 Bioanalyzer and was finally quantified by qRT-PCR. According to the quantification results and the final sequencing data amount, the sequencing library mixed different samples were immersed in the sequencing process and were denatured by 0.1 M NaOH to generate single-stranded DNA. Then the single-stranded DNA was amplified using TruSeq SR Cluster Kit v3-cBot-HS (#GD-40103001, Illumina) in situ. The libraries mixed different samples were sequenced by Illumina HiSeq 4000 (service provided by Kangchen Biotech, Shanghai, China) by running 150 cycles.

The LG samples from type 2 diabetic mice or vehicles were collected with Trizol reagent. The mRNA was enriched by NEBNext^® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), and the produced RNA was used for construction Library, via KAPA Stranded RNA-Seq Library Prep Kit (Illumina). The prepared RNA-seq libraries were qualified using Agilent 2100 Bioanalyzer and quantified by the qPCR absolute quantification method. The sequencing was then performed using Illumina NovaSeq 6000. After quality control, the fragments were 5′, 3′-adaptor trimmed and filtered ≤ 16 bp reads with cutadapt software. The trimmed reads were aligned to a reference genome (source: genecode, version: GRCm38) with Hisat2 software. The transcript abundance for each sample was estimated with StringTie (v1.2.3), and the Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) values for gene-level were calculated with R package Ballgown (v2.6.0). The differentially expressed genes (DEGs) analysis was also performed with Ballgown. Fold change (cutoff 1.5), p-value (cutoff 0.05), and FPKM (≥0.5 mean in one group) were used for filtering DEGs and transcripts. GO analyses of DEGs were performed via DAVID (6.8, http://david.ncifcrf.gov)(Gene Ontology, 2021 (link)). Gene count > 2 and p < 0.05 were set as the threshold.

To investigate the target genes regulated by FTSJ1, PC9 cells were transfected with FTSJ1 or scrambled controls for 48 h. Total RNA was isolated from PC9 cells using the TRIzol (Invitrogen, Shanghai, China) reagents, and RNA quantity and quality were measured using the NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Shanghai, China). mRNA was pulled down using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, MA, USA). RNA-seq libraries were prepared using the KAPA Stranded RNA-Seq Library Prep Kit (Illumina, CA, USA), and sequencing was performed on an Illumina HiSeq 4000 by KangChen Biotech Company (Shanghai, China). Sequencing reads were trimmed using StringTie and mapped to human genome database (GRCh37) by the Hisat2 software. Differential expression (FPKM, fragments per kilobase of gene/transcript model per million mapped fragments) were calculated using the Ballgown software. The sequencing data set has been deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE146604.

RNA sequencing was performed using the 6G RNA Sequencing Service (150bp paired-end, 40 million reads) (Arraystar Inc., Rockville, MD, USA). In brief, mRNA was enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs Inc., Ipswich, MA, USA) and RNA sequencing library preparation was performed using a KAPA Stranded RNA-Seq Library Prep Kit (Illumina, San Diego, CA, USA). Sequencing (150 cycles for both ends) was performed on an Illumina Novaseq 6000. Image analysis and base calling was performed using Solexa pipeline v1.8 (Off-Line Base Caller software, v1.8) and sequence quality was examined using FastQC software. Trimmed reads (trimmed 5’, 3’-adaptor bases using cutadaptor) were aligned to the reference genome (GRCm38) using Hisat2 software. The transcript abundances for each sample were estimated using StringTie [45 (link)] and the FPKM, differential gene expression and volcano plots were generated using R package Ballgown [12 (link)]. RNA sequencing data are available at Gene Expression Omnibus (accession no. GSE144796).