Blood and cell culture dna mini kit

DNA was purified for sequencing from cells infected in six-well plates using a blood and cell culture DNA minikit (Qiagen). Samples were sequenced at the Northwestern University Genomics Core Facility using NextGen Illumina HiSeq SR500 sequencing. Unipro UGene (57 (link)) was used to create a trimmed reference sequence composed of the HSV-1 F strain genome (GenBank accession number GU734771.1) with the long- and short-terminal repeat regions deleted (58 (link)). The gB (UL27) gene in the reference sequence was replaced with either the SaHV-1 gB sequence or the KOS strain gB sequence (GenBank accession number KT899744.1) with the gB3A substitutions added. Using Geneious 8.1.9, sequencing reads were aligned to the reference genome and variants were identified. Variants present in both the revertant viruses and the original BACs were ignored, as were variants in the gJ gene due to the RFP insertion. Mutations identified in other glycoproteins are reported in Table 1. The SaHV-1 gB sequence of HSV-SaHVgB^pass viruses was confirmed using standard single pass DNA sequencing after PCR amplification.

TZM-bl cells were seeded in 96-well flat-bottom plates. The NT assay was performed the next day when the monolayer was ~80% confluent. The reaction mixture contained 10 µL (5 µg/mL or 20 µg/mL) of mAb 2F5, 5 µL of normal human serum, and different amounts of p48 KYNU (or rKYNU). The mixture was adjusted to 50 µL with complete DMEM (TZM-bl cells) or RPMI 1640 (Jurkat cells). HIV-1_NL4-3 (initial infectious titer 2 × 10⁵/mL) was diluted in complete DMEM to gain the MOI 0.5 and added to each reaction mixture. Incubation was performed for 1 h at 37 °C and afterwards, the mixtures (100 µL) were loaded on TZM-bl cells. After 48 h cells were washed twice with PBS and fixed with 2.5% paraformaldehyde for 15 min and washed in PBS. The luminescence was measured using the Bright-Glo™ Luciferase Assay System (Promega, Madison, WI, USA) and the signal was estimated using GloMax^®-96 microplate luminometer (Promega, Mannheim, Germany). The NT assay on Jurkat cells was performed as described above, but 2 × 10⁵ cells were used for infection, and dilutions were prepared using a complete RPMI 1640 medium. 48 h post-infection the Jurkat cells were harvested and washed twice with PBS. Extraction of DNA was performed using Blood and Cell Culture DNA Mini kit (Qiagen GmbH, Hilden, Germany). Proviruses were quantified by qPCR using the HIV-1 gag detection system.

Two female adult L. elegans spiders were obtained from Binzhou county, Dali City, Yunnan province, China in 2021. The live samples were sent to Beijing Biomarker Technologies for sample handling, DNA and RNA extraction, and sequencing. Briefly, the spiders were cleansed and grinded in liquid nitrogen, respectively. One spider was used for Hi-C sequencing and RNA sequencing (RNA-seq). The other was for genome sequencing, including next-generation and Nanopore sequencing. Genomic DNA was extracted using a Blood and Cell Culture DNA Mini Kit (Qiagen, Germany) according to the protocol. The short paired‐end insert libraries, including Hi-C and genome sequencing, were constructed using the Illumina platform protocol, and 150-bp paired-end reads were generated using the Illumina NovaSeq platform (Illumina NovaSeq 6000 Sequencing System, U.S America RRID:SCR_020150). A genome long read library was constructed and sequenced on the Nanopore Oxford platform (Oxford Nanopore Technologies, England, RRID: SCR_003756). Total RNAs of the whole body were extracted using TRIzol (Invitrogen, U.S America) according to manufacturer instructions and RNA-seq was generated on the Illumina NovaSeq platform.

Genomic DNA was isolated using Qiagen’s blood and cell culture DNA mini kit (Qiagen, Valencia, CA) according to the protocol given in the technical manual. DNA concentration and purity of the isolated genomic DNA sample were measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Genomic DNA sample was separated on a 1% agarose gel and the quality of the sample was visualized by imaging the gel with a BioRad Molecular Imager Gel Doc XR+ (BioRad, Hercules, CA).
Forward and reverse 16S rRNA PCR primers were designed based on sequences in the literature for selecting primer pairs with the best overall coverage and phylum spectrum to reduce the bias in PCR-based microbial diversity studies
^{48 (link)}. The sequence of the forward primer 16SF is: 5’ CCTACGGGNGGCWGCAG 3’ and that of the reverse primer 16SR is: 5’ GACTACHVGGGTATCTAATCC 3’. HotStar Taq Plus DNA polymerase enzyme kit (Qiagen, Valencia, CA) was used for PCR following the given cycling conditions given in the Qiagen protocol booklet.