Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

We use RNA-seq to study the transcriptome wide specificity profiles of A-to-I RNA editing by split-ADAR2 constructs. All RNA-seq experiments were carried out in HEK293FT cells which were grown in DMEM supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher) in an incubator at 37 °C and 5% CO2 atmosphere. Plasmid transfection for the split-ADAR2 samples was carried out according to standard protocols. RNA was extracted using the RNeasy mini kit. RNA-seq libraries were prepared from 250ng RNA using the NEBNext Ultra RNA Library Prep Kit for Illumina. Basecalls were performed using Illumina RTA3, and fastq files were then generated using Illumina bcl2fastq2 v2.20. Both tasks were carried out by the UCSD IGM Genomics Center. RNA-seq read pairs with 100 bases per read mate were aligned to the GRCh38 reference genome using STAR aligner version 2.7.1a or 2.7.3a (Dobin A et al 2013). The genome index was built using primary assembly annotations from GENCODE release 32. Default parameters were used to run STAR, except for the following relevant settings: --clip3pAdapterSeq AGATCGGAAGAGCACACGTCTGAACTCCAGTCA, AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT, --outSAMtype BAM Unsorted, --outBAMcompression 0, --quantMode GeneCounts, --outReadsUnmapped Fastx, --genomeLoad LoadAndRemove, --alignSJDBoverhangMin 1, --peOverlapNbasesMin=10, --peOverlapMMp=0.05, --outSAMmultNmax 1, --alignEndsType EndToEnd, --outFilterMismatchNmax -1, --outFilterMismatchNoverReadLmax 0.2, --outFilterMultimapNmax 1. The reads of the resulting uniquely aligned pairs were sorted by genomic coordinate using samtools sort (Li H. et al 2009). Duplicated read pairs were marked using samtools markdup and were removed from subsequent RNA-editing analysis. The uniquely aligned reads for each sample were down-sampled using samtools view with option -s. Down-sampling fractions were calculated by dividing the smallest number of uniquely aligned reads among all samples by the number of uniquely aligned reads available for the sample being down-sampled. Down-sampling was not performed on the reads of the control samples. The down-sampled reads where then fed to samtools mpileup, whose output was parsed to extract the counts of each base found at each A-site and T-site in the GRCh38 reference genome. Insertions and deletions were ignored. Reference sites with read coverage of less than 10 were discarded. From the remaining reference A- and T-sites a final list was selected by choosing sites common to all samples and with at least one G or C at a reference A- or T-site, respectively, in at least one sample. For each pair of compared control and treatment samples, and for each reference A- or T- site selected as described above, a Fisher exact test was carried out to calculate the p-value for the change in proportion of G or C bases, respectively, at that site. The p-values for all selected reference sites were adjusted for multiple testing using the Benjamini-Hochberg method. The measured base proportions for each site (row) and sample (column) are reported in processed data file pm_adar_rned_008-mcplns_seq_rna_ed_bfscr_0_res-bpr-0.tsv.gz. The adjusted p-values for the comparison (column) of base proportion between each sample and the control sample at each site (row) are reported in processed data file pm_adar_rned_008-mcplns_seq_rna_ed_bfscr_0_res-pad-0.tsv.gz.