Hifiscript gdna removal cdna synthesis kit

Total RNA was isolated from different samples using the RNA-Solv^® reagent (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. RNA concentrations were determined using a NanoDrop 2000 UV Spectrophotometer (Thermo Fisher Scientific, Cheshire, UK). The genomic DNA removed with (10× gDNA Remover Mix) and the first strand of cDNA was synthesized using a HiFiScript gDNA Removal cDNA Synthesis Kit (Cwbiotech, Taizhou, China) according to the manufacturer’s protocol, followed by storage at −80 °C until use.

To validate the NGS data, qRT-PCR was used to confirm the expression levels of several modulated miRNAs. The Mir-X™ miRNA First-Strand Synthesis Kit (TaKaRa, Dalian, China) synthesized the miRNA’s first-strand cDNA. The U6 snRNA was used as the internal reference gene. For mRNA quantification, the HiFiScript gDNA Removal cDNA Synthesis Kit (CWBIO, China) was used to synthesize first-strand cDNA. GAPDH was used as the internal reference gene. The relative expression values were normalized by the internal control. Quantitative real-time PCR analysis was performed using LightCycler^®96 (Roche Diagnostics GmbH, Germany) with TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) Kit (Takara, Dalian, China), following the manufacturer’s instructions. Triplicate qRT-PCR was performed on each cDNA sample to guarantee the reproducibility of the amplification. After amplification, the relative fold change of the differentially expressed miRNAs and mRNA were calculated through the 2^−ΔΔCT method [49 (link)]. The primers were designed using the Oligo 7.0 software and they are listed in Table S8. TSINGKE Biololgical Technology (China) synthesized all of the primers used in this study.

Total RNA and miRNA from HK-2 cells and tissue were isolated using a miRNA Purification Kit (CWBio, China). The miRNA was reverse-transcribed into cDNA with a miRNA cDNA Synthesis Kit (CWBio, China). The mRNA was reverse-transcribed into cDNA using a HiFiScript gDNA Removal cDNA Synthesis Kit (CWBio, China). The mRNA and miRNA levels were tested by qRT-PCR using UltraSYBR Mixture (Low ROX) (CWBio, China). The expression of TGF-β, α-SMA, and 3-phosphoinositide-dependent protein kinase 1 (PDPK1) mRNA was normalized to that of the GAPDH gene, and U6 was used as the internal reference for mir-129-5p. The primer sequences used for qRT-PCR are shown in Table 1. The fold change was calculated using the 2^−∆∆Ct method, and the means from three independent experiments were used.

Total RNA was isolated with the OmniPlant RNA Kit (DNase I) CW2598 (CWBIO, Beijing, China) as previously described [45 (link)]. HiFiScript gDNA Removal cDNA Synthesis Kit CW2582 (CWBIO, Beijing, China) was used to achieve first-strand cDNA synthesis from approximately 1 µg of total RNA. qRT–PCR was performed using the iQ SYBR Green Supermix (Bio–Rad, Hercules, CA, USA) and run on the ABI Prism 7000 system (Applied Biosystems, Foster City, CA, USA). The sequences of the genes studied in this article (BrActin, Bra022356; BrPAL, Bra005221; BrC4H, Bra018311; Br4CL, Bra030429; BrCHS, Bra008792; BrCHI, Bra007142; BrF3H, Bra036828; BrF3’H, Bra009312; BrFLS, Bra009358; BrDFR, Bra027457; BrANS, Bra013652; BrLDOX, Bra019350; BrUFGT, Bra023954) were derived from a previously published paper [36 (link)] and Brassica database BRAD (http://brassicadb.cn, accessed on 20 January 2022) [46 (link)]. Furthermore, BrActin2 was used as the reference housekeeping gene. The relative expression level of the target genes was normalized against the reference housekeeping gene [47 (link)]. The primers used in the qRT–PCR are listed in Supplementary Table S2.

Overnight cultured strains were inoculated in 25 mL M9 medium in a 1% inoculum. After incubation to logarithmic growth phase at 30°C and 150 rpm, the cells were collected for mRNA extraction and processed according to the manual with an RNA pure Bacteria Kit (CWbiotech, Beijing, China). Genomic DNA was then removed for reverse transcription and synthesis of cDNA using the HiFiScript gDNA Removal cDNA Synthesis Kit (CWbiotech). Real-time quantitative polymerase chain reaction(RT-qPCR) was performed using ultra SYBR mixtures (CWBiotech) on a CFX96 touch RT-qPCR detection system (Bio-Rad, Hercules, CA, USA). 16S rRNA was amplified using RT-16S-F/RT-16S-R primers and used as an internal reference. The primer pairs of RT-sadA-F/RT-sadA-R, RT-sadB-F/RT-sadB-R, RT-sadC-F/RT-sadC-R, RT-ssuE-F/RT-ssuE-R, RT-rutF-F/RT-rutF-R, RT-cogA-F/RT-cogA-R, RT-sutR-F/RT-sutR-R, and RT-psuK-F/RT-psuK-R were used to amplify sadA, sadB, sadC, ssuE, rutF, cogA, sutR, and psuK, respectively. Relative transcript levels of these genes were calculated using the 2^−∆∆CT method (31 (link)), with P. denitrificans DYTN-1 as the control.

Total RNA was isolated from pig tissues using Trizol reagent (Invitrogen, USA). The HiFiScript gDNA Removal cDNA Synthesis Kit (CWBiotech, Beijing, China) was sued for cDNA synthesis. Q- PCR was conducted using the 2 × plus SYBR real-time PCR mixture (BioTek, Beijing, China) on a QuantStudio 3 system (Thermo fisher, USA). The primer sequences are listed in Table S1. Relative gene expression levels were calculated using the 2^-ΔΔCt method with GAPDH as the internal control.

Skin tissue (obtained in D11 and D23, mentioned in Section 4.3) of the rat was removed at −80°C and weighed at 50–100 mg by the analytical balance in an RNA enzymes free tube (2 mL) and treated with 1 mL TRIzol Reagent. Total RNA was extracted with RNA Extraction Kit (Solarbio, Beijing) according to the manufacturer's protocol, and quantified by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA).
Following the manufacturer's process, one microgram of RNA was reverse transcribed by the HiFiScript gDNA Removal cDNA Synthesis Kit (CWBIO). Several transcripts were measured using PerfectStar Green qPCR SuperMix (+Dye I/+Dye II) (TRANS, China) through ViiA™ 7 Real‐Time PCR System (Applied Biosystems). Specific primers are listed in Table 1.