Dna clean concentrator 25

In collaboration with Prof. Peter Girguis's Laboratory (Department of Organismic and Evolutionary Biology, Harvard University), the total RNA from gill samples was DNAse-digested using the TURBO DNA-free™ Kit (Ambion® Life Technologies). Reverse-transcription was performed using the SuperScript™ III First-Strand Synthesis System (Invitrogen™, ThermoFisher Scientific) with random hexamers priming for first-strand synthesis (50 ng/µl, provided in the SuperScript™ III First-Strand Synthesis System), followed by second-strand synthesis using the SuperScript Double-Stranded cDNA Synthesis Kit for (Invitrogen™ ThermoFisher Scientific). cDNA was purified with the QIAquick PCR Purification Kit (Qiagen; Valencia, CA) and quantified using the Qubit® Fluorometer (Life Technologies). The samples were then sheared into approximately 400 bp fragments using the Covaris S220 ultrasonicator (Covaris; Woburn, MA), with the following parameters: duty cycle, 10%; intensity, 4; 200 cycles per burst; 72 sec. Sheared DNA fragments were concentrated using the DNA Clean & Concentrator™-25 (Zymo Research; Irvine, CA) and quantified using Qubit® Fluorometer (Life Technologies).

Genomic sites of interest were amplified into fragments of approximately 200 bp from genomic DNA samples using PrimeSTAR GXL DNA Polymerase (TaKaRa). See Supplementary Table 3 for the list of primers used and the average mapped ratio of corresponding primers. PCR products were purified using DNA Clean & Concentrator-25 (Zymo Research) for Sanger sequencing and targeted deep sequencing. Targeted deep sequencing libraries were prepared using the VAHTS Universal DNA Library Prep Kit for Illumina v.3 (Vazyme). Briefly, the PCR fragments were sequentially subjected to end repair, adapter ligation and then PCR amplification. DNA purification in library preparation was performed using Agencourt Ampure XP beads (Beckman Coulter), and library amplification was performed using Q5U Hot Start High-Fidelity DNA Polymerase (NEB) and VAHTS Multiplex Oligos Set 4/5 for Illumina (Vazyme). The final library was subjected to quantification using the Qubit dsDNA HS Assay Kit (Invitrogen) and sequenced using Illumina HiSeq X Ten.

NGS of the virus populations was carried out as described^{47 (link),48 (link)}. Briefly, RNA was extracted with Trizol LS and the RNA Clean & Concentrator-5 (Zymo research) kit. The reverse transcription was carried out with Maxima H Minus Reverse Transcriptase (ThermoScientific), the whole open reading frame was amplified with polymerase chain reaction (PCR) Q5 Hot start High-Fidelity DNA Polymerase (New England Biolabs), and the PCR product was purified (DNA Clean & Concentrator-25 and Zymoclean Large Fragment DNA Recovery Kit, Zymo research). The NEBNext ultra II FS DNA Library Prep Kit (New England Biolabs) was used for library preparation, and sequencing was performed with an Illumina Miseq platform.
The alignment of amino acid sequences of structural proteins (Core, envelope proteins E1 and E2) of 1a and 5a HCV was done in BLAST (database version 5; https://blast.ncbi.nlm.nih.gov/Blast.cgi)^{49 (link),50 (link)}.

Nuclei (between 50,000 and 60,000) were used to isolate RNA using Single-Cell RNA Purification Kit (Norgen, catalog# 51800). In brief, aliquots of nuclei were resuspended in 350 µl of RL buffer (Norgen) and passed through an 18 G syringe five times. RNA extraction, including DNase digestion, followed manufacturers’ instructions. RNA was eluted in 20 µl of Elution Solution A (Norgen). The nuclear RNA concentration was determined using TapeStation (Agilent). RNA was diluted to 1 ng/µl and a total of 5 ng was processed for RNA-seq library preparation. RNA libraries were prepared using NuGen Ovation RNA-Seq System V2 (#7102–32) for cDNA preparation following the product manual. cDNA purification was done using Zymo Research DNA Clean & Concentrator-25 with modification from the Ovation protocol. cDNA, eluted in 30–40 µl of TE (1 µg per sample), was fragmented at 300 bp using Covaris S2 (Sonolab S-series V2), followed by library preparation according to KAPA LTP Library Preparation Kit (KK8232), using Illumina indexed adapters. Libraries were sequenced on NovaSeq 6000.

Extracted nucleic acids from individual macrophyte species were pooled by spring site, resulting in the following four pools: Blue (12 species), Ichetucknee (11 species), Manatee (six species), and Rainbow (16 species) (Table 1). Pooled nucleic acids were reversed-transcribed for two independent NGS efforts (Table 2). Reverse transcription was performed with the Superscript IV First-Strand Synthesis System for RT-PCR (Invitrogen, Waltham, MA, USA) using random hexamers provided by the manufacturer or a random primer tagged with a known linker sequence following manufacturer’s protocols. Products from the former were used without pre-amplification for NGS library preparation (cDNA libraries), whereas products from the latter were used for sequence-independent, single-primer amplification (SISPA libraries, see below) (Table 2). Reverse-transcribed products obtained with random hexamers for cDNA libraries were subjected to second-strand synthesis with the Klenow Fragment DNA Polymerase (New England Biolabs, Ipswich, MA, USA). For cDNA libraries, 80 µl of double-stranded reverse-transcribed product were prepared from each spring site pool and purified with either Agencourt AMpure XP beads (Beckman-Coulter, Brea, CA, USA) or the DNA Clean & Concentrator®-25 (Zymo Research, Irvine, CA, USA) for fragmentation and NGS library preparation (Table 2).