GSM9579693: HEK293T, RiboScan Reporter, Library 4, sublibrary 6, rep2; Homo sapiens; OTHER
Genomic DNA was extracted from sorted cell populations using the GeneJET genomic DNA extraction kit (ThermoFisher). For transcription start site mapping, total RNA was extracted using the RNeasy kit (Qiagen) from cells expressing the RiboScan library. Test sequences from each sorted FACS gate were PCR amplified using Q5 high-fidelity DNA polymerase with high-GC buffer for 20–25 cycles. Reverse primers contained an 8-nt barcode corresponding to the FACS gate identity (See supplementary file "Barcodes_and_GFP_intensities_per_sublibrary_per_gate.xlsx" for an overview of the applied barcodes). PCR products were gel-purified and quantified using a Qubit dsDNA high-sensitivity assay. Amplicons were pooled at equimolar ratios. Illumina sequencing adapters were added using TruSeq small RNA primers in a final 8-cycle PCR. Each biological replicate was assigned a unique TruSeq index.  5′ Rapid Amplification of cDNA Ends (5′RACE) was performed to determine transcription start sites (TSSs) of RiboScan reporters. Total RNA was extracted from cells expressing the RiboScan reporter library using the RNeasy Kit (Qiagen). First-strand cDNA synthesis was carried out with a primer complementary to the GFP sequence in the RiboScan reporter construct (5′-TCTTGTAGGTCCCGTCGTCT-3′), using SuperScript III reverse transcriptase (Invitrogen) for 45 minutes at 55 °C. RNA was subsequently removed by adding 1 µL of RNase Cocktail Enzyme Mix (ThermoFisher) per 20 µL of reaction volume. cDNA was purified using AMPure XP beads (Beckman Coulter) at a 0.8x bead-to-sample ratio. The purified cDNA was A-tailed using terminal deoxynucleotidyl transferase (TdT; NEB) in the presence of a 1000-fold molar excess of dATP over the 3′ ends, resulting in A-tails of approximately 10–20 nucleotides. After a second bead cleanup (0.8x ratio), second-strand synthesis was performed using primer 5′-TTCAGAGTTCTACAGTCCGACGATCNNNNNNCCTTTTTTTTTTTTTTTTTV-3′, containing a six nucleotide long unique molecular identifier (UMI). Following another bead cleanup, PCR amplification (20 cycles) was performed using forward primer 5′-TTCAGAGTTCTACAGTCCGACGATC-3′ and reverse primer 5′-GTGACTGGAGTTCCTTGGCACCCGAGAATTCCAATGTTCAGGTGCCCGTTGACGTCGC-3′, with complementarity to the GFP sequence in the RiboScan reporter. Illumina index 1 and index 2 adapters were added in a final PCR (8 cycles).
GEO
GSM9579693_r1