High fidelity 2 pcr master mix

PCR was performed with NEBNext® High-Fidelity 2× PCR Master Mix (M0541). Thermal cycler parameters were set to: initial denaturation for 5 min at 98 °C, 20 cycles of denaturation at 98 °C for 30 s, annealing for 30 s at 58 °C, extension for 40 s at 72 °C, and final extension for 5 min at 72 °C. PCR products were purified via 1% agarose gel electrophoresis and QIAquick Gel Extraction Kit (Qiagen, 28706). All samples were denatured and diluted according to the Illumina NextSeq system denature and dilute libraries guide (document # 15048776 v09, illumina.com) and sequenced on an Illumina NextSeq 500. Custom Python scripts, cutadapt 2.8 and Bowtie2 2.3.0 were used to deconvolute the raw data and determine the abundance of individual gRNAs in each sample^{53 (link),54 (link)}

HEK293FT cells were transfected as described with 25 ng of a plasmid encoding a dCas13bt3-ADAR2dd(E488Q) fusion expressed from a CMV promoter, 300 ng of a plasmid encoding a crRNA expressed from a human U6 promoter, and 45 ng of a dual Gaussia/ Cypridina(W85X) luciferase reporter plasmid (Cox et al., 2017 (link)) (Table S1). After 48 h, the RNA was harvested, and reverse transcription was then performed as described (Joung et al., 2017 (link)), using a gene-specific primer for the Cypridian luciferase (5′-TTTGCATTCATCTGGTACTTCTAGGGTGTC-3′). cDNA was used as input for the preparation of next-generation sequencing libraries with NEBNext High-Fidelity 2 × PCR Master Mix (NEB), and amplicons were then sequenced on an Illumina MiSeq. Editing was quantified by counting the number of reads at which the expected edited position in the amplicon was identified as G and dividing by the total number of reads in the sample, using Python. Unless otherwise noted, all reported data are the average of four biological replicates. To measure the restoration of the Cypridina luciferase (W85X) activity, the culture media were aspirated from the same cell samples and the Cypridina and Gaussia luciferase activities in the media were measured as described above.

A nontemplated-addition reverse transcription reaction (no TSO present; 50-μl total volume) contained 0.1 μm RNA template (5′-OH, 5′-p, or 5′-m⁷G–capped 4rN-25mer-RNA-FAM), 1× Template Switching RT Buffer, 1 mm dNTP solution mixture, 30 nm i7 primer, and 500 units of Template Switching RT. The reaction was performed at 42 °C for 90 min followed by a 10-min heat-denaturation step at 72 °C. After the reaction, 2.5 units of RNase H (catalog number M0297) and 25 units of RNase I_f (catalog number M0243) were added to the reaction and incubated at 37 °C for 20 min to hydrolyze the RNA template. The cDNA was purified using an Oligo Clean & Concentrator. The purified cDNA was incubated in a 20-μl reaction with 750 nm 5′-AppDNA (see supporting information for sequence), 1× NEBuffer 1, 5 mm MnCl₂, and 2 μm Thermostable 5′-AppDNA/RNA Ligase (catalog number M0319) at 65 °C for 60 min to perform 3′ single-strand cDNA ligation. The reaction was inactivated at 90 °C for 3 min. The ligated products were subjected to PCR amplification with NEBNext Universal and Index primers (catalog number E7335) using NEBNext High-Fidelity 2× PCR Master Mix (catalog number M0541). The libraries were cleaned up with NEBNext Sample Purification Beads (catalog number E7767) and sequenced on an Illumina iSeq-100 as described above.

CUT&Tag assays were performed as described previously with some modifications (50 (link)). Briefly, 1 × 10⁵ cells were washed twice with wash buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine and 1× protease inhibitors] by gentle pipetting. A 10 μl aliquot of concanavalin A-coated magnetic beads (Bangs Laboratories, BP531) was activated and added per sample and incubated at RT for 10 min. The supernatants were removed and bead-bound cells were resuspended in 100 μl of wash buffer containing 0.01% digitonin and 2 mM EDTA. The H3K9me2 antibody (Cell Signaling Technology, 4658) was added and incubated overnight at 4°C on a rotator. The beads were washed and resuspended in 300-wash buffer containing 300 mM NaCl with pG-Tn5 (Vazyme, S602) at RT for 1 h. The beads were washed and tagmentation was performed in 300-wash buffer supplemented with 10 mM MgCl₂ at 37°C for 1 h. To stop the tagmentation reaction, 2.25 μl of 0.5 M EDTA, 2.75 μl of 10% SDS and 5 μl of proteinase K (20 mg/ml) were added and further incubated at 55°C for 2 h. Then the genomic DNAs were extracted by phenol–chloroform and subjected to PCR amplification using NEBNext High-Fidelity 2× PCR Master Mix (NEB, M0541S) for 13 cycles. The libraries were size-selected with 1.2× AMpure XP beads (Beckman Coulter, A63881) and sequenced on a NovaSeq 6000 sequencer.