Longamp taq 2 master mix

The SalFAD3.LA1 and SalFAD3.LA2 allele-specific markers were generated using primer pair No 12 (Table S1) which was designed based on the conserved flanking sequences of intron 3. The PCR reaction was performed with LongAmp Taq 2× Master Mix (NEB) following the manufacturer’s instructions with a 60°C annealing temperature.

The H9c2 cell line was purchased from the cell bank of the Institute of Biochemistry and Cell Biology of Shanghai (Shanghai, China) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin streptomycin (Gibco, Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazoliumbromide (MTT) was bought from Sigma Aldrich Inc. (St Louis, MO, USA). Doxorubicin and luminescence-enhanced ATP assay kit were purchased from Beyotime Biotech Inc. (Shanghai, China). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was obtained from Thermo Fisher Scientific Inc. (Rockford, IL, USA). DNA extraction kit (TIANGEN, Beijing, China), Long Amp Taq 2× Master Mix (NEB, Ipswitch, MA, USA) and Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) were used for mitochondrial DNA damage assay. Primary antibodies for p-JNK, JNK, Mfn1, and Mfn2 were purchased from Abcam Inc. (Cambridge, MA, USA). Primary antibodies for β-actin and caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA), HRP goat anti-rabbit antibody and HRP goat anti-mouse antibody were purchased from Beijing BioDee Biotechnology Co., Ltd. (Beijing, China).

Fecal samples were used for total RNA extraction using a miRNA isolation kit with phenol (Cat#AM1561, Invitrogen). Multiplex Small RNA Library Prep Set (NEBNext) were used to construct the small RNA-seq libraries. PCR amplification were performed using LongAmp Taq 2× Master Mix (Cat#M0287L, NEB) and quality was determined on an Agilent Bioanalyzer 2100 system. Then, the library preparations were constructed on an Illumina Hiseq 2500 platform, All the 18 ~ 26 nucleotide-length unique sequences were mapped to specific species precursors in miRBase 22.0. Different miRNA expression was analyzed using Student t test.

Library preparation for cDNA-PCR sequencing was performed using the SQK-PCS109 sequencing kit, following the manufacturer’s instructions (Oxford Nanopore Technologies, Oxford, UK). Briefly, 50 ng of enriched microbial RNA samples from L93, L96, and L101 were reverse transcribed using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA) and incubated in a thermal cycler at 42 °C for 90 min and then 85 °C for 5 min. To amplify the products without introducing biases, full-length transcripts were selected using the Nanopore rapid attachment primers (SQK-PCS109) using LongAmp Taq 2× Master Mix (New England Biolabs, Ipswich, MA, USA) with the following cycling conditions, an initial denaturing step at 95 °C for 5 min, followed by 14 cycles of 15 s at 95 °C, 15 s at 62 °C, and 1 min at 65 °C, and a final extension step at 65 °C for 6 min and a hold at 4 °C. The products were then purified and separated using Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN) with 1.8 (v/v) ratio of magnetic beads to the reaction mixture. The cDNA library using 100 fmol of amplified cDNA with adapters was loaded onto a FLO-MIN106D R9 flowcell and sequencing was performed with a MinION device for 48 h. cDNA derived from each replicate (L93, L96, L101) were sequenced on individual flowcells to provide information about the process reproducibility and variability.

Library construction, sequencing, and data analysis were entrusted to Novogene (Tianjin, China). Sequencing libraries were generated using NEBNext Multiplex according to the manufacturer’s recommendations. The small RNA (sRNA) molecules were ligated to a 5′ adaptor and a 3′ adaptor using T4 RNA ligase 1. Then, first-strand cDNA was synthesized using M-MuLV reverse transcriptase. PCR amplification was performed using LongAmp Taq 2× master mix (NEB, USA). At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High-Sensitivity Chips (Agilent Technologies, USA). After cluster generation, the library preparations were sequenced on an Illumina HiSeq 2500/2000 platform, and 50-bp single-end reads were generated.
Raw reads were first processed through custom perl and python scripts. In this step, clean reads were obtained by removing low-quality reads. The small RNA tags were mapped to reference sequence by Bowtie without mismatch to analyze their expression and distribution on the reference. Using miRBase (http://www.mirbase.org) as reference, modified software miRDeep2 was used to obtain the potential miRNA, and miREvo and miRDeep2 were integrated to predict novel miRNA (79 (link), 80 (link)). Expression analysis of miRNAs was performed using the same method as used for circRNAs.

A library of plasmids harboring his3 and ura3 expressed under the control of randomized 28-mers was constructed based on published methods (36 (link)). The strand complementary to a commercial 71-mer oligonucleotide was synthesized by PCR. NotI restriction fragments were separated on a 20% polyacrylamide gel. The larger band was excised and digested with EcoRI. Final purification by a Oligo Clean & Concentrator kit retained the desired sticky-ended 28-mer library but not the 6-nucleotide by-product. The insert was ligated into pH3U3-mcs, and the resulting library was transformed directly into the counterselection strain. We performed counterselection three times using liquid gel medium instead of solid agar (37 (link)). The final plasmid library was tested using pB1H2w2-mutOdd and pB1H2w2-Zif268 control plasmids.
The omega subunit of RNAP was fused to full-length RR_1586 and three constructs of its DNA-binding domain (at positions Arg124, Ser131, and Gln151) using sequence- and ligation-independent cloning (38 (link)). PCR amplification of vector pB1H2w2-Prd (Long-Amp Taq 2× master mix; NEB) and the insert introduced complementary overhangs to be recombined in vitro by RecA. Plasmid construction was confirmed by Sanger sequencing. Plasmid DNA from overnight cultures in 50 ml of LB was isolated and concentrated by ethanol precipitation in preparation for selection (36 (link)).