Zymo clean and concentrator kit

Whole-genome sequencing was performed on a subset of convenience samples at the California Academy of Sciences’ Center for Comparative Genomics (https://www.calacademy.org). cDNA was purified by using the Zymo Clean and Concentrator Kit (Zymo Research, https://www.zymoresearch.com). Library preparation was performed by using the Nextera XT DNA Library Preparation Kit (96 samples) and the Nextera XT Index Kit (96 indices, 384 samples) (both from Illumina, https://www.illumina.com) according to manufacturer’s instructions with modifications: 3 ng of input cDNA with 4 μL index was used per reaction (rather than the suggested 1 ng input with 5 μL index because of low cDNA concentrations). Samples were tagged with a molecular identification tag. Libraries were quantified and assessed for quality on a BioAnalyzer (Agilent, https://www.agilent.com) before equimolar pooling for sequencing on the Illumina MiSeq platform using reagent run kits in series Nano, V2, and V3. Raw reads were uploaded by using CLC Genomics Workbench version 7.0.3 (https://www.qiagen.com) after being sorted by sample. DENV sample reads were mapped to complete, reference genome sequences downloaded from GenBank for each serotype. Virus consensus sequences were extracted in CLC Genomics Workbench version 7.0.3 and exported for phylogenetic analyses.

Plant and bacterial DNA were extracted as previously published [29 (link)]. Forward and reverse primers were designed to anneal to genomic DNA sequences at approximately 250 bp flanking the putative clipping sites (S2 Table). PCR was performed using PfuUltra II Hotstart 2X Master Mix (Agilent Technologies, Inc). After amplification the PCR products were purified using the Zymo Clean and Concentrator Kit (Zymoresearch, USA). For direct sequencing of PCR products, the same primer set for amplifications were used. For sequencing of cloned PCR products, PCR fragments were cloned directly into pCR2.1 using the manufacturer’s recommendations for the Zero Blunt Topo cloning kit (Life Technologies), and sequenced using M13F and M13R primers. In both cases DNA traces were compared to wild type sequences using the SeqMan function from LaserGene analysis package. For directly sequenced PCR products, deviations from a consensus trace that originated near the -3 position relative to the NGG PAM site, the known site of Cas9 cutting, were scored as evidence of genome editing in somatic tissues relative to wild type traces.

Extracted gDNA was sent to Vanderbilt Technologies and Advanced Genomics Facility, Nashville, TN. The library was prepared using TruSeq DNA Sample Preparation Guide v2, catalog #FC-930-1021 (Illumina, San Diego, CA, USA). One g of gDNA was sheared using a Covaris S2. Sheared ends were then repaired and adenylated and the products were ligated with Illumina adaptors. Ligated fragments were then size selected using Pippen Prep (Sage Science, Beverly, MA, USA) and cleaned using Zymo Clean and Concentrator Kit (Zymo Research, Irvine, CA, USA). KAPA Hot Start (KAPA Biosystems, Wilmington, MA, USA) was then used the amplify libraries over a total of 14 PCR cycles. Clustering was performed using a cBot (Illumina) and paired-end sequencing was performed on a HiSeq 2000 (Illumina) over two lanes.

A 5′ rapid amplification of cDNA ends (RACE) was performed according to a protocol provided by Life Technologies. Briefly, total RNA was isolated from S. aureus UAMS-1 and AH1263 cultures grown for 6 h (late exponential growth phase) in low-oxygen conditions using the RNeasy Mini Kit (Qiagen) as previously described (32 (link)). nos cDNA was then generated using the iScript Select cDNA Synthesis Kit (Bio-Rad) and the primer nos-GSP1 (Table S2). cDNA was purified using the Zymo Clean and Concentrator kit (Zymo Research) and used in a homopolymeric tailing reaction with 2 mM dCTP and 15 units of TdT (Invitrogen). The reaction product was amplified further in several rounds of PCR using nested gene-specific primers (nos-GSP2 and nos-GSP3; Table S2), and products were visualized on a 1% agarose gel using gel electrophoresis. Once a suitable amount of DNA was visualized, products were sent for Sanger sequencing (Genewiz) with primers nos-GSP3 and nos-screen (Table S2). The TSS (or the first nucleotide of the mRNA) was then determined from sequencing results by locating the homopolymeric tail and the first nucleotide following it. Similar methods were used with lacZ-specific primers (lacZ-GSP1, lacZ-GSP2, lacZ-GSP3, and lacZ-screen; Table S2) to map the TSS in nos-lacZ fusion plasmids pJBnos1 and pJBnos2. Sanger sequencing results are provided in Supplementary file 1.

Biopsy samples were homogenized with Precellys 25 system with zirconium oxide beads (Bertin Technologies SAS, Montigny-le-Bretonneux, France). Following homogenization, RNA was extracted using the total RNA extraction protocol, with the RNeasy kit (QIAGEN Pty Ltd., Melbourne, VIC, Australia). After DNAse treatment, using TURBO DNAse I, each sample was purified using the Zymo Clean and Concentrator Kit as per the manufacturer’s instructions (Zymo Research, CA, United States). The RNA concentration was measured by a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States). Samples without the optimal 260:280 ratio, which was between 1.8 and 2.1, were excluded from the experiment. The RNA integrity was verified by Agilent Bioanalyzer (Agilent, Santa Clara, CA, United States), and only samples with an RNA integrity number (RIN) above eight (RIN > 8) were used for RNA sequencing. When needed, RNA extraction was repeated to achieve this quality and integrity.