Preparation of genomic DNA and RNA amplicons for HTS
This protocol is extracted from research article:
Analysis and minimization of cellular RNA editing by DNA adenine base editors
Sci Adv, May 8, 2019; DOI: 10.1126/sciadv.aax5717

A two-step PCR protocol was performed as previously reported (1). Briefly, 1 μl of isolated genomic DNA was input into the first round of PCR (PCR1). Phusion U Multiplex Master Mix (Thermo Fisher Scientific) was used for both PCR steps. PCR1 was performed with the primers listed in the Supplementary Materials for the appropriate sgRNA treatment for 30 cycles with an annealing temperature of 61°C and an extension time at 72°C for 15 s. Upon verification that PCR1 was successful by running the products on a 2% agarose gel, the barcoding PCR (PCR2) was set up using primers to incorporate barcodes for Illumina sequencing. All primers were ordered from Integrated DNA Technologies. After PCR2, up to 240 samples with different barcode combinations were combined and purified by gel extraction using the QIAquick Gel Extraction Kit (QIAGEN). A second column was used for full removal of agarose and ethidium bromide before the product was quantified using the Qubit ssDNA HS Assay Kit (Thermo Fisher Scientific) and sequenced using an Illumina MiSeq with 220- to 260-bp single-end reads.

For RNA, primers were used as listed in the Supplementary Materials to amplify the targeted region of cDNA. Quantitative PCR (qPCR) was used for all experiments to avoid overamplification of the cDNA. RSL1D1 required more PCR cycles (34) than IP90 and CTNNB1 (32 each) using the program: 98°C for 1 min and 30 s, then cycles of (98°C for 10 s, 60°C for 15 s, and 72°C for 15 s), followed by a final extension of 2 min at 72°C. No reverse transcriptase controls and no input controls were also processed by qPCR and carried forward onto the MiSeq for each experiment. In no instances did either control exceed 2.5% of the number of aligned reads for the particular experiment when compared to the corresponding RNA samples.

For assessing the number of adenosines within an amplicon that showed greater than 0.1% editing, the percentage of G for each adenosine position was measured and counted in Microsoft Excel using the formula = COUNTIF(C85:HS85,“>0.001”), where C85:HS85 represents the range of cells containing the frequency of bases called as a guanosine when the interrogated nucleoside is an adeosnine (for nonadenosine positions, the value within the C85:HS85 range is set to zero).

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