Hindiii hf

Tissue samples were collected from young leaves, freeze-dried, and pulverized using GenoGrinder 2000 (Spex CertiPrep Inc. Metuchen, NJ, USA) at 650 strokes/min for 15 s. Genomic DNA was isolated using 96-well silica filter plates (Epoch Life Science #2020-001). DNA was quantified using PicoGreen (Thermo Fisher Scientific, Waltham, MA) on a Synergy™ HT plate reader (Bio-Tek Instruments, Winooski, VT, USA) and diluted to 50 ng/µl in EB buffer. Genomic libraries were constructed using the two-enzyme GBS approach^{12 (link)} using simultaneous restriction-ligation with HindIII-HF, MseI, and T4 DNA Ligase (New England Biolabs). Restriction-ligation reactions were pooled by 96-well plate, purified using AMPure XP beads, PCR-amplified using Phusion Master Mix (NEB #M0531), and purified again using AMPure XP beads. A DNA7500 chip in an Agilent 2100 Bioanalyzer was used to determine average library size and concentration, and to dilute each library to 10 nM before submission for sequencing on an Illumina Hiseq 2500 (SR100) at the UC Davis Genome Center (Davis, CA).

RNA was isolated from glomeruli using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen). cDNA was synthesized from 100 ng of RNA using the High Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher Scientific). Multiplexed qPCR was performed on a QuantStudio 6 Flex Real-Time PCR System with TaqMan Gene Expression Master Mix (4370048) and gene expression assays (APOL1: Hs01066280_m1; IRF1: Mm01288580_m1; GAPDH: Mm99999915_g1; Thermo Fisher Scientific). Relative quantification was performed using the delta-delta Ct method. Allelic discrimination assay was performed as previously described, but with cDNA instead of gDNA (Ruchi et al., 2015 (link)). Briefly, PCR was performed on 10 ng cDNA using APOL1-specific primers (F- 5′-GCCAATCTCAGCTGAAAGCG–3′; R: 5′-TGCCAGGCATATCTCTCCTGG-3′). Purified PCR product (Cat. no. 28106, Qiagen) was digested using HindIII-HF or NspI (R3104 and R0603, New England Biolabs) and run on a 2% agarose gel.

pCAGGS.SARS-CoV-2_SΔ19_fpl_mNG2(11)_opt was generated using NEBuilder DNA assembly (New England Biolabs) of a pCAGGS vector backbone cleaved using EcoRV-HF and HindIII-HF (New England Biolabs) and a PCR fragment encoding a codon-optimized SARS-CoV-2 Wuhan-Hu-1 spike protein (amplified from pCMV3-C-Myc; VG40589-CM, SinoBiological) with a C-terminal 19 amino acid deletion as described in (Ou et al., 2020 (link)). A 12 amino acid flexible protein linker (fpl) and a modified 11^th betasheet of mNeonGreen (Feng et al., 2017 (link)) were added at the C-terminus. pCAGGS.BSD_fpl_mNG2(11) was generated using NEBuilder DNA assembly of a pCAGGS vector backbone cleaved using EcoRV-HF and HindIII-HF and the Blasticidin S deaminase gene (BSD) PCR amplified from a pLenti6.3 vector. Afterwards, cDNA encoding for a 12-amino acid fpl and a modified 11^th betasheet of mNeonGreen were inserted at the 3´end of the insert ORF. pcDNA3.1.mNG2(1–10) was generated through NEBuilder DNA assembly of a pcDNA3.1 vector (Thermo Fisher Scientific), amplified by PCR, and 10 betasheets of a modified mNeonGreen synthesized by Genscript. For pCAG3.1/SARS2-Sd19 PCR-amplified Wuhan-Hu-1 spike sequence (from pCMV3-C-Myc) was inserted via blunt end cloning in the pCAG3.1 acceptor vector cut with EcoRV-HF.

Genotyping‐by‐sequencing libraries were prepared by restricting genomic DNA with HindIII‐HF (NEB) with Ampure XP bead (Beckman‐Coulter) cleanup. Sequence data were obtained on an Illumina Hiseq 4000 instrument at the UC Davis Genome Center. Single nucleotide polymorphism (SNP) calling was performed using the TASSEL GBS pipeline (Glaubitz et al., 2014 (link)) with alignment to the Chandler genome (Marrano et al., 2020 (link)), resulting in 13,320 polymorphic SNPs. The phangorn package in R (R Core Team, 2017 ; Schliep et al., 2017 ) was used to construct a neighbour‐joining tree from the distance matrix.

To cut out the transgene from the plasmid backbone, a restriction digest reaction was set up as a 50 μL reaction in a PCR tube as follows:

Reagent	Volume/concentration
Restriction enzyme (HindIII-HF, NotI-HF, New England Biolabs)	1 μL of each
10X CutSmart® buffer (New England Biolabs)	5 μL
Plasmid DNA	1 μg
dH2O	Remaining volume (up to 50 μL)

The reaction was run for four hours at 37 °C on a thermocycler

End resection was assayed in ER-AsiSI cells that were predominantly in S/G2 phase by cell synchronization procedure mentioned earlier in the Methods section. The extent of resection adjacent to specific DSBs was measured by quantitative polymerase chain reaction (qPCR) as described previously [24 (link)]. The sequences of qPCR primers are shown in S1 Table. 20μL of genomic DNA sample (~200 ng in 1x CutSmart NEB restriction enzyme buffer) was digested or mock digested with 20 units of restriction enzymes (NmeAIII, AvaI, BsrGI, BamHI-HF or HindIII-HF; New England Biolabs) at 37°C overnight. 5 μl of digested or mock digested samples (~20 ng) were used as templates in 20 μl of qPCR reaction containing 10 μl of 2x iTaqUniversal SYBR Green Supermix, 500 nM of each primer using iCycler iQReal-Time PCR (Bio-Rad). The % ssDNA generated by resection at selected sites was determined as previously described [24 (link)]. Briefly, for each sample, a ΔCt was calculated by subtracting the Ct value of the mock-digested sample from the Ct value of the digested sample. The % ssDNA was calculated using algorithm: ssDNA% = 1/(2^{^}(ΔCt-1) + 0.5)*100 [24 (link)]. Data represent the mean of at least three independent experiments with SD values indicated by error bars.