Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: Total DNA and RNA of transfected cells were extracted by the Qiagen ALLPrep DNA/RNA Mini Kit following the manufacturer's instruction Extracted RNA was subjected to DNase treatment in a 375 L system with 2.5 L Turbo DNase, and 37.5 L Turbo DNase Buffer at 37°C for 1 hr. 3.75 L 10% SDS and 37.5 L 0.5M EDTA was added to stop DNase with 5 mins of incubation at 75°C. The whole volume was used for eGFP probe hybridization in an 1800 L system, with 450 l 20X SSC Buffer, 900 L Formamide, and 1 L of each 100 M Biotin-labeled GFP probe One to Three. The probe hybridization was performed through incubation at 65°C for 2.5 hrs. 200 l Dynabeads™ MyOne™ Streptavidin C1 was prepared according to the manufacturer's instructions. The beads were suspended in 250 L 20X SSC Buffer and incubated with the above probe hybridization reaction at room temperature for 15 mins. Beads were then collected on a magnet and washed with 1X SSC Buffer once, and 0.1X SSC Buffer twice. eGFP mRNA was eluted first through adding 12.5 L ddH2O, heating at 70°C for 2 mins and collecting on a magnet, then adding another 12.5 L ddH2O, heating at 80°C for 2 mins and collecting on a magnet. All collected elution was performed with another DNase treatment in a 30 L system containing 0.5 L Turbo DNase, and 3 L Turbo DNase Buffer at 37°C for 1 hr. 0.5 L 10% SDS was added to halt DNase reactions. Eluted mRNA was purified through RNA Clean SPRI Beads. mRNA was reverse transcribed to cDNA using SuperScript™ IV First-Strand Synthesis System with specific primer MPRA_v3_Amp2Sc_R, following the manufacturer's instructions. cDNA, extracted DNA and plasmid control were then used for building sequencing libraries following Tag-seq Library Construction section in the paper of Tewhey et al (PMID: 27259153). A total of two PCR rounds were needed for building the sequencing library. The first PCR was performed with TruSeq_TruSeq_Universal_Adapter_P5 and MPRA_V3_Illumina_GFP_F. The second PCR was performed with TruSeq_TruSeq_Universal_Adapter_P5 and index primer. Samples were purified, molar pooled and sequenced using Illumina 1x100bp on Illumina Novaseq 6000