title: GSM6119040: PRO-seq SW480 Rep2; Homo sapiens; OTHER
library_construction_protocol: Nuclei from SW480 cells were isolated and snap-frozen in storage buffer [10 mM tris-HCl (pH 8.0), 25% glycerol, 5 mM MgCl2, 0.1 mM EDTA, and 5 mM dithiothreitol (DTT)]. Next, 10 × 1M nuclei and 100 l of storage buffer were run on by the addition of 100 l of prewarmed 2× reaction buffer [10 mM tris-HCl (pH 8.0), 300 mM KCl, 5 mM MgCl2, 1% sarkosyl, 1 mM DTT, RNaseOUT (0.8 U/l; ThermoFisher Scientific), 250 M adenosine 5-triphosphate,250 Mguanosine 5-triphosphate,50 M biotin-11cytidine 5-triphosphate (PerkinElmer), and 50 M biotin-11uridine 5-triphosphate (PerkinElmer)]for 3 min at 37°C. Reactions were stopped by the addition of 500 l of TRIzol LS (Invitrogen) and purified following the manufacturers instructions. RNAs were heat-denatured at 65°C for 40s, placed on ice, and fragmented with 0.2 N of NaOH on ice for 10 min. After pH adjustment with 1M tris-HCl (pH 6.8), buffer exchange using P-30 columns (Bio-RadLaboratories) was performed. The 3 RNA adaptor (VRA3:5-GAUCGUCGGACUGUAGAACUCUGAAC-/inverted dT/-3) was ligated to the eluted RNAs, and a second biotin RNA enrichmentwas performed as described above. Next, the 5 end of RNA swas enzymatically modified with RNA 5 pyrophosphohydrolase(NEB), and hydroxyl repair was performed using T4 polynucleotidekinase (NEB). Purified RNAs were subsequently ligated with the 5RNA adaptor (RA5: 5-CCUUGGCACCCGAGAAUUCCA-3).A third biotin RNA enrichment was performed before reverse transcription of the eluted RNAs. Libraries from two biological replicates were amplified for 13 cycles, size-selected for 140 to 350 bp, and paired-end sequenced on Illumina HiSeq 4000 (75 bp).