GSM5920176: MPRA mRNA rep 1; Homo sapiens; OTHER
GSM5920176
Total RNA was extracted from cells using QIAGEN Maxi RNeasy (QIAGEN, 75162) following the manufacturer's protocol including the on-column DNase digestion. A second DNase treatment was performed on the purified RNA using 5 μL of Turbo DNase (Life Technologies, AM2238) with buffer, in 750 μL of total volume for 1 h at 37 °C. The digestion was stopped with the addition of 7.5 μL 10% SDS and 75 μL of 0.5 M EDTA followed by a 5-min incubation at 70 °C. The total reaction was then used for pulldown of GFP mRNA. First-strand cDNA was synthesized from the DNase-treated GFP mRNA with SuperScript III. Single-stranded cDNA was purified by SPRI and eluted in 30 μL EB. To minimize amplification bias during the creation of cDNA tag sequencing libraries, samples were amplified by qPCR to estimate relative concentrations of GFP cDNA using 1 μL of sample in a 10 μL PCR reaction containing 5 μL Q5 NEBNext master mix, 1.7 μL SYBR Green I diluted 1:10,000 (Life Technologies, S-7567) and 0.5 μM of TruSeq_Universal_Adapter and MPRA_Illumina_GFP_F primers. Samples were amplified with the following qPCR conditions: 95 °C for 20 s, 40 cycles (95 °C for 20 s, 65 °C for 20 s, 72 °C for 30 s), 72 °C for 2 min. The number of cycles for sample amplification was 1−n (the number of cycles it took for each sample to pass the threshold) from the qPCR. To add Illumina sequencing adapters, 10 μL of cDNA samples and mpra:minP:gfp plasmid control (diluted to the qPCR cycle range of the samples) were amplified using the reaction conditions from the qPCR scaled to 50 μL, excluding SYBR Green I. Amplified cDNA was SPRI purified and eluted in 40 μL of EB. To determine oligo/barcode combinations within the MPRA pool, Illumina libraries were prepared from the plasmid library by performing five separate amplifications with 200 ng of plasmid in a 100 μL Q5 NEBNext PCR reaction.  Amplified material was SPRI purified using a 0.6 × bead/sample ratio and eluted with 30 μL of EB. Sequencing indexes were then attached using 20 μL of the eluted product and the same reaction conditions as for the tag-seq protocol, except the number of enrichment cycles was lowered to 5. Samples were molar pooled and sequenced using 2 × 150 bp chemistry on an Illumina NextSeq 550.