title: GSM9002449: HCT 116 FLAG/FKBPF36V-DNTTIP1, 2h dTAGv1, rep1; Homo sapiens; OTHER
library_name: GSM9002449
library_construction_protocol: Cells were washed x2 ice cold PBS and scraped into ice-cold permeabilisation buffer (10mM Tris/Cl pH 8, 10m KCl, 250mM sucrose, 5mM MgCl2, 1mM EGTA, 0.1% IGEPAL CA-630, 0.5mm DTT, 0.05% Tween-20, 10% glycerol, PIC and SUPERase-In RNase inhibitor). Cells were transferred to a falcon and incubated on ice for 5min. Permeabilised cells were collected by centrifugation at 700 rcf for 4min at 4C, washed twice (permeabilisation buffer without IGEPAL CA-630 and Tween-20) and resuspended in freeze buffer (50mM Tris/Cl pH 8, 40% glycerol, 5mM MgCl2, 1.1mM EDTA, 0.5mM DTT, SUPERase-In RNase inhibitor). Cells were counted, collected by centrifugation at 1000 rcf for 5min at 4C and resuspended freeze buffer at 1x10^6 cells/52ul for aliquoting and snap freezing. The run-on reaction was performed by mixing 50ul permeabilised cells 1:1 with 2x run-on master mix (10mM Tris/Cl pH 8, 5mM MgCl2, 1mM DTT, 300mM KCl, 40uM each of Biotin-11-(CTP/UTP/ATP/GTP), 1% sarkosyl, SUPERase-In RNase inhibitor) and incubating at 37C, 750rpm for exactly 5min and stopped by the addition of RL buffer. RNA was extracted using a Norgen RNA extraction kit (Norgen) and 1M NaOH used to hydrolyse bases. RNA was precipitated using ethanol and resuspended in  nuclease-free water. The purified RNA was used to anneal a 3' adapter (/5Phos/rUrNrNrNrNrNNGATCGTCGGACTGTAGAACTCTGAAC/3InvdT/) before streptavidin bead binding, on-bead 5' hydroxy repair, on-bead 5' decapping. 5' adaptor ligation (/5InvddT/CCTTGGCACCCGAGAATTCCANrNrNrNrNrNrC) was performed on bead and RNA purified using TRIzol. Reverse transcription was performed using with RP1 primer (AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA). Test amplification was performed to calculate the required number of cycles for full-scale amplification using a single Illumina indexing primer (CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA). Full-scale amplification was performed using 1 of 4 Illumina indexing primers for each sample (CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA; CAAGCAGAAGACGGCATACGAGATAGATCGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA; CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA; CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA). DNA was purified after full-scale amplification using AMPure beads, quantified using the qubit HS dsDNA assay and profiled using a bioanalyzer.
identifiers.namespace: GEO
identifiers.id: GSM9002449_r1