GSM8156041: experiment: mRNA, plate_number: 1, [DMS]:1.5%, replicate: A; synthetic construct; OTHER
For the in vitro transcription of the human mRNA library, all the designed amplicons were amplified via PCR, adding a T7 promoter sequence at the forward primer (5′ TAATACGACTCACTATAG 3′ ) using a 2X PCR PreMix (Syd Labs, Cat. MB067-EQ2N), human cDNA (ZYAGEN, Cat. HD-UR-40) and an INTEGRA VIAFLO384 channel pipette. For each 384 plate, 5 µl of each individual reaction were pooled together and purified using a Zymo DNA clean up kit (Cat. D4003). The PCR product pool was used as template for the in vitro transcription, using the T7 MEGAscript kit (Thermo Fisher, Cat. AMB13345) according to the manufacturer's instructions. After DNase treatment, the transcribed RNAs were purified using a Zymo RNA clean up kit (Cat. R1017). 10 µg of the purified RNAs were diluted in 10 µl of nuclease free water, denatured for 1 minute at 95 degrees and placed on ice for 2 minutes. 88.5 µl of the refolding buffer (sodium cacodylate 397.5 mM and MgCl2 6 mM final) were added and the RNAs were left refolding at 20 minutes at 37ºC. After, 1.5 µl of DMS (dimethyl sulfate) was added to obtain a 1.5% final concentration, for 5 minutes at 37ºC, while shaking at 600 RPM. The reaction was stopped by adding 60 µl of β-mercaptoethanol, followed by Zymo RNA clean up.    Libraries were generated as previously described and the indexed samples were sequenced in a Illumina NextSeq 2000.    For the pri-miRNA library, in vitro transcription templates were prepared via 8 cycles of PCR using 2x Q5 Master Mix (New England Biolabs, Cat. M0492S) with a T7 promoter containing forward primer as previously described. Following column clean up (Zymo Research, Cat. D4013), 100 ng of template DNA was used in a 2 hour in vitro transcription reaction using the HiScribe T7 High Yield RNA (New England Biolabs, Cat. E2040S) kit according to the manufacturer's instructions. Following DNase digestion (Invitrogen, Cat. AM2238) and column clean up (Zymo Research, Cat. R1017), 1 ug of RNA from each library was DMS treated separately as described above, substituting a 1.5% final DMS concentration for 2.5%. Following DMS treatment, the samples were reverse transcribed with Induro Reverse Transcriptase (New England Biolabs, Cat. M0681L) for 30 minutes at 55°C using the appropriate reverse primer for each library according to the manufacturer protocol. After alkaline hydrolysis with 1 ul of 4M NaOH, cDNA was cleaned and concentrated using Zymo Oligo Clean and Concentrator (Zymo Research, D4060). Sub-library specific primers were then used to amplify 1 ul of cDNA template for 24 cycles using Advantage 2 PCR Mix (Takara Bio, Cat. 639206). Finally, library indexing was carried out with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Cat. E7645S) and sequenced on a NextSeq 1000 P2 cartridge (Illumina, Cat. 20046813)  in accordance with the manufacturer's instructions. To generate the human mRNA library, all human protein-coding transcripts were downloaded from GENCODE Release 43 [CITE GENCODE]. Every transcript with a non-AUG start codon or without an annotated 5' UTR, CDS, and 3' UTR was discarded. To focus on 3' UTRs and maximize the likelihood of successful RT-PCRs, for each gene, the longest common suffix (i.e. subsequence beginning at the 3' end) among all isoforms was selected. Each gene was discarded if it had no annotated name, if its longest common suffix was shorter than 253 nt, or (to minimize mispriming and misalignment) if its longest common suffix contained any 41-mers that were present in the longest common suffix of any other gene. To verify that alignment to the wrong gene would be minimal, a simulated FASTQ file containing every 128 nt segment in 64 nt increments from every gene's longest common suffix was aligned to all longest common suffixes with Bowtie2 v2.5.1 [CITE Bowtie2].  Primers for human mRNAs were designed against the longest common suffixes of all remaining genes using Primer3 v2.6.1 [CITE Primer3] with the parameters PRIMER_TASK=generic, PRIMER_PICK_LEFT_PRIMER=1, PRIMER_PICK_INTERNAL_OLIGO=0, PRIMER_PICK_RIGHT_PRIMER=1, PRIMER_PRODUCT_SIZE_RANGE=253-1021, PRIMER_OPT_SIZE=28, PRIMER_MIN_SIZE=20, PRIMER_MAX_SIZE=36, PRIMER_WT_SIZE_LT=0.0, PRIMER_WT_SIZE_GT=0.0, PRIMER_MIN_TM=65.0, PRIMER_OPT_TM=75.0, PRIMER_MAX_TM=80.0, PRIMER_PAIR_MAX_DIFF_TM=10.0, PRIMER_GC_CLAMP=0, PRIMER_MAX_NS_ACCEPTED=0, P3_FILE_FLAG=0, PRIMER_EXPLAIN_FLAG=1, SEQUENCE_OVERHANG_LEFT=TAATACGACTCACTATAGGG. A T7 promoter ending in three G bases was prepended to every forward primer. Genes that received no primer pairs were discarded; for those that received more than one pair, the pair that would produce the longest amplicon was selected. The sequences of the amplicons (including only the last 3 Gs of the T7 promoter) were used as the reference sequences in the subsequent analysis.  To select amplicons for verification in HEK 293T cells, four microarray datasets of gene expression in HEK 293T cells from two studies [CITE Alla et al. and Abramson et al.] were downloaded from the National Center for Biotechnology Information's Gene Expression Omnibus [CITE NCBI GEO] (accession numbers GSM389756, GSM389757, GSM609202, and GSM609205). Microarray probe IDs were converted to gene names using DAVID [CITE DAVID]. The amplicons of at most 600 nt (the maximum paired-end read length on Illumina sequencers) were ranked by the minimum expression level of their genes among the four datasets (to be conservative); the 96 highest-ranked amplicons were chosen for mutational profiling in HEK 293T cells.  To generate the pri-miRNA library, human genome annotations were downloaded from miRBase (mirbase.org, Kozomara A, Birgaoanu M, Griffiths-Jones S Nucleic Acids Res (2019) 47:D155-D162; Kozomara A, Griffiths-Jones S Nucleic Acids Res (2014) 42:D68-D73; Kozomara A, Griffiths-Jones S Nucleic Acids Res (2011) 39:D152-D157; Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ Nucleic Acids Res (2008) 36:D154-D158; Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ Nucleic Acids Res (2006) 34:D140-D144, Griffiths-Jones S Nucleic Acids Res (2004) 32:D109-D111). After removing redundant sequences, 1292 miRBase hairpins were selected for downstream library creation. The miRBase hairpins were then padded with their flanking genomic sequence to a total of 192 nt using Biopython's Entrez interface [CITE Biopython, NCBI Entrez]. These 1292 sequences were then separated into 3 sub-libraries to reduce jackpotting during library processing. To each construct, a sub-library specific pair of 19 nt universal primer binding regions were added to minimize primer associated PCR and reverse transcription bias, bringing the full length library size to 230 nt. The final sequences were then synthesized as an oligo pool (Agilent Technologies).