Qubit high sensitivity dna kit

The SARS-CoV-2 genome of thirteen samples was enriched using ARTIC primer scheme version 3; information on the primer sequences and protocols are available at the ARTIC network repository (https://artic.network/ncov-2019).
The Nextera DNA Flex Library Preparation kit (Illumina, USA) with dual indexing was used for library preparation. The libraries were purified with AMPure XP beads (Beckman Coulter, USA). All quantifications were carried out using a Qubit DNA High-Sensitivity kit (Invitrogen, USA). The library’s control quality and length were assessed on a Fragment Analyzer 5200 system (Agilent Technologies, USA) using the Standard Sensitivity NGS Analysis Kit (Agilent Technologies, USA).
Whole-genome sequencing was performed on an Illumina MiniSeq (Illumina, USA) platform using MiniSeq^TM Mid Output Reagent Cartridge (300-cycles, paired-end reads). Adapter/quality trimming and filtering raw data were performed with BBDuk, and clean reads were mapped to the consensus genome using Geneious Prime 2020.1.2 (https://www.geneious.com).

RNA was extracted from CCA samples using a modified TRIzol® RNA extraction protocol from Invitrogen. CCA samples were removed from RNAlater and then homogenised in Trizol (1 mL) at room temperature for 6 mins at 30 Hz using a QIAgen TissueLyser. After the initial 3 mins of homogenisation, samples were removed, placed on ice for 5 mins, and then homogenised for the remaining 3 mins. Further processing followed the manufacturer’s protocol, using bromochloropropane for phase separation, and high-salt solution for precipitation. RNA pellets were resuspended in DNase/RNase-free distilled water (20 µl). Total RNA quantity was determined spectrophotometrically using Invitrogen Qubit® Broad Range RNA kit. RNA yield ranged from 5.36 ng RNA/µl to 800 ng RNA/µl. Presence of contaminating DNA was checked randomly in samples using an Invitrogen Qubit® DNA High Sensitivity kit and returned readings “too low for detection”.

All samples used for genomic DNA extraction required for the study of in vivo STR polymorphism of pfcyp19b promoter region were derived from TRACII clinical trials samples collected at patients’ admission. gDNA used for TRAC2 amplicon sequencing was extracted from TRIzol (Invitrogen) fractions after the removal of total RNA. In brief, Buffer containing 4M guanidine thiocyanate (Sigma), 50mM sodium citrate (Sigma) and 1M Tris (BioRad) was mixed with organic TRIzol fraction (bottom phenol phase) and incubated at room temperature for 10 minutes with continuous agitation and subsequently centrifuged at 10000 x g. The aqueous phase containing solubilized genomic DNA was aliquoted from the top fraction and treated with RNAse A (Qiagen) and Proteinase K (Qiagen). 100% ethanol was added, and the mixture was subsequently transferred onto QIAamp DNA Blood column (Qiagen) and gDNA was extracted following manufacturer’s protocol. Eluted genomic DNA was vacuum desiccated at room temperature and resuspended in nuclease-free water. DNA samples were quantified with Qubit DNA High Sensitivity kit (Invitrogen) and stored at -20°C.
gDNA used for in vitro transfection validation and qPCR analysis was extracted using Quick-DNA kit (ZYMO) following manufacturers instruction.

Polyclonal DNA minipreps isolated from the third panning output pools were used as PCR template to amplify the HCDR3 of the selected Fabs and add the adapters required for sequencing on Illumina sequencer MiSeq. The PCR protocol has been described (Liu et al, 2019).
The PCR product was purified and DNA concentration was measured using the Qubit DNA High sensitivity kit (Invitrogen). Samples were analyzed on a MiSeq using MiSeq reagent kit MiSeq v2 Reagent kit 300 cycles PE.

PCR was performed using GoTaq DNA Polymerase (Promega, Madison, WI) with primers listed in Supplementary file 1. PCR products were electrophoresed using 1% agarose gels in sodium boric acid buffer. Following electrophoresis, gels were dyed with GelRed (Phenix Research, Candler, NC) and imaged on an Alpha Innotech GelRed Imager (Alpha Innotech, San Leandro, CA). Amplified bands were excised from the gels and purified with an SV Wizard Gel Cleanup kit (Promega). Following purification, DNA concentration was measured using the Qubit DNA high sensitivity kit (Life Technologies, Grand Island, NY) and sequencing reactions were performed by Genewiz (South Plainfield, NJ).