title: GSM4725182: SMIT Nab2-AA 10 min; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Nascent RNA was extracted as previously described (Carrillo et al 2010). Briefly, chromatin isolated from frozen cell pellets is incubated with phenol-chloroform to extract nucleotides. DNA is digested with DNase and polyA sequences are depleted using oligo(dT) beads. For long read sequencing, ribosomal RNA was depleted using a cap-dependent exonuclease. SMIT library construction was performed as described previously (Carrillo et al. 2016). Briefly, a 3′ end adapter (/5rApp/NNNNNCTGTAGGCACCATCAAT/3ddC/) was ligated to nascent RNA and samples were reverse transcribed. Gene-specific forward primers were used to amplify the cDNA and a second PCR added Illumina library adapters. Long read sequencing utilized the same adapter ligation strategy used in SMIT. Strand-switching reverse transcription created full length double-stranded cDNA molecules with common 5′ and 3′ ends. An initial PCR of 12-15 cycles amplified product for input into the ONT PCR Barcoding kit (SQK-PBK004) which includes dA-tailing and barcode adapter ligation. A final PCR of 8-10 cycles enriched for successful ligation and AMPure XP bead purification removes products under 250 bp. The final library was loaded onto an Oxford Nanopore MinION flow cell and sequenced for 48 hours.