Agencourt ampure xp pcr purification beads

The V3-V4 hypervariable regions of the 16S rDNA gene were amplified with the forward primers 341F (5’-CCTACGGGNGGCWGCAG-3’) and the reverse primer 805R (5’-GACTACHVGGGTATCTAATCC-3’). For fungi, the internal transcribed spacer (ITS) regions were amplified with the primer ITS1 (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS2 (5’-GCTGCGTTCTTCATCGATGC-3’) [58 (link)]. The PCR components were as follows: 10× Toptaq buffer (1 µL), Toptaq DNA Polymerase (0.2 µL), 2.5 mM dNTPs (0.8µL), 10 µM of each Forward and Reverse primer (0.2 µL), DNA Template (1 µL), and up to 10 µL of ddH₂O. The total volume of the reaction was 10 µL. Amplification conditions consisted of an initial denaturation step at 94 °C for 2 min, followed by 25 cycles consisting of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min, with a final extension of 10 min at 72 °C. PCR amplicons were purified with Agencourt AMPure XP PCR Purification Beads (Beckman Coulter, Indianapolis, USA) and quantified using the Invitrogen Qubit3.0 Spectrophotometer Kit (Thermo Fisher Scientific, CA, USA). The PCR products were sequenced using the Illlumina NovaSeq platform with NovaSeq Reagent Kit v3 at Genesky Biotechnology Co., Ltd. (Shanghai, China).

For RNA sequencing 100,000 fluorescent liver cells were sorted directly to TRIzol LS (Thermo Fisher Scientific, USA). After ethanol precipitation RNA was depleted of DNA by using DNase I treatment and purified on columns by using RNA Clean & Concentrator™-5 (Zymo Research, USA). RNA integrity was measured by RNA ScreenTape on the Agilent 2200 TapeStation system (Agilent Technologies, USA). RNA Integrity Number (RIN) was in the range from 8.5 to 10 for all the samples used for RNA-seq. Ribosomal RNA removal from 10 ng of total RNA was performed using RiboGone Kit (Clontech Laboratories, USA). cDNA synthesis for next-generation sequencing (NGS) was performed by SMARTer Universal Low Input RNA Kit (Clontech Laboratories, USA) as recommended by the manufacturer. DNA libraries were purified with Agencourt AMPure XP PCR purification beads (Beckman Coulter, USA) and DNA fragment distribution was assessed by using D1000 ScreenTape and Agilent 2200 TapeStation system (Agilent Technologies, USA). KAPA library quantification kit (Kapa Biosystems, USA) was used for qPCR-based quantification of the libraries obtained. Paired-end sequencing (2 × 75 bp reads) was performed with NextSeq 500 sequencing system (Illumina, USA).

For ATAC-seq 60,000 fluorescent liver cells were sorted to Hank’s solution (1× HBSS, 2 mg/mL BSA, 10 mM Hepes pH 8.0), centrifuged for 5 min at 500×g and prepared for chromatin tagmentation as previously described [80 (link)]. NEBNext High-Fidelity 2 × PCR Master Mix (New England Biolabs, USA) and custom HPLC-purified primers containing Illumina-compatible indexes were used to prepare DNA sequencing libraries as previously described [81 (link)]. DNA libraries were purified with Agencourt AMPure XP PCR purification beads (Beckman Coulter, USA) and DNA fragment distribution was assessed by using D1000 ScreenTape and Agilent 2200 TapeStation system (Agilent Technologies, USA). KAPA library quantification kit (Kapa Biosystems, USA) was used for qPCR-based quantification of the libraries obtained. Paired-end sequencing (2 × 75 bp reads) was performed with NextSeq500 sequencing system (Illumina, USA).

Total DNA was extracted from approximately 0.20 g of soil collected on days 0, 14, and 28 and frozen at −80°C by using the DNeasy PowerSoil Kit (Qiagen, Germany). DNA concentrations were determined with a Nanodrop 2000c spectrophotometer (Thermo, USA). Then, the DNA was stored at −20°C for further high-throughput sequencing.
The following primers were used to amplify the V4-V5 region of the bacterial 16S rRNA gene: 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′). The 50-μL PCR mixture contained 2 ng of soil DNA, 25 μL TopTaq DNA Polymerase (Transgen, China), 1.0 μL of each primer and double-distilled water to adjust the final volume. The PCR was carried out on an ABI 2720 Thermal Cycler (Applied Biosystems, USA), and the PCR products were purified with Agencourt AMPure XP PCR Purification Beads (Beckman Coulter Ltd., UK). The purified PCR products were sequenced on an Illumina NovaSeq 6000 system (Genesky Biotechnologies Inc., China) according to the manufacturer’s recommendations.

During the polymerase chain reaction, which was conducted with a TopTaq DNA polymerase kit (Transgen, Beijing, China), the V3–V4 regions from the bacterial 16S rDNA gene were amplified with universal primer set 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) [41 (link)]. According to the manufacturer’s protocol, final PCR products were purified and quantified by Agencourt AMPureXPPCR Purification Beads (Beckman Coulter, Grants Pass, OR, USA) and Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The sequencing was performed on Illumina NovaSeq 6000 platform (2 × 250 bp) at Genesky Biotechnologies Inc. (Shanghai, China). Amplicon sequencing is the high-throughput sequencing of PCR products of specific gene segments such as 16S rDNA, 18S rDNA, and ITS [42 (link)]. Among them, the 16S rDNA sequencing used in this study is an important and widely used tool for obtaining information about microbial communities’ structures, microbial taxonomy, and community diversity [43 (link)].

Total RNA was extracted with TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed by Genesky Biotechnologies Inc. (Shanghai, P.R. China). Briefly, total RNA quality was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The mRNA were fragmented and purified using AgencourtAMPure XP-PCR Purification Beads (Beckman Coulter, Brea, CA, USA). Double size selection was performed using AgencourtSPRIselect Reagent Kit (Beckman Coulter). A Qubit and Agilent 2100 Bioanalyzer was used to check the quality of the short fragment library; RNA-sequencing was performed on an Illumina HiSeq platform (Illumina, San Diego, CA, USA) with 2 × 150 bp. The raw sequencing reads were evaluated by FastQC (V0.11.4) The KEGG and GO enrichment analysis was performed as previously described [30 (link)].