title: GSM8514644: TREx-BCBL1 Rta cell with 24hr lytic KSHV, biological repeat control 1; Homo sapiens; RIP-Seq
library_construction_protocol: After 24 hours of lytic replication, cells were fixed with DSP (0.5 mg/mL) for 45 minutes at room temperature, washed three times with 20 mM Tris in PBS (pH 7.5), then permeabilised with 0.25% Triton in PBS for 15 minutes at room temperature. Cells were washed twice, followed by the addition of 6 mL of diluted probe mixture (25 nM DIG-labelled NEAT1 oligonucleotides in buffer (2xSSC buffer, 10% formamide, 10% dextran sulphate)). A control condition was included, in which 6 mL of buffer was added to cells with the absence of the probes. The cell culture plates were incubated at 37 °C overnight in a humidified chamber.  Samples were washed before being blocked, then incubated in purified HyPro protein (2.7 ng/mL) before washing again. Biotin-phenol and hydrogen peroxide were introduced to the cells for 1 minute followed by immediate quenching. Cells were lysed in high-SDS RIPA lysis buffer before scraping into a 1.5 mL microcentrifuge tube for further lysis. Samples were sonicated for 7 cycles of 30 seconds ON/ 30 seconds OFF, incubated at 37 °C for 30 minutes, then incubated in proteinase K. RNA was extracted from the lysates using TRIzol LS (Invitrogen) as per the manufacturers protocol and 5% from each sample were retained for inputs. MyOne streptavidin C1 pulldowns were performed on the remaining 95% of samples and final purified biotinylated RNA was extracted via the TRIzol LS extraction method coupled with the RNA clean and concentrator kit (Zymo Research). Library construction was carried out as per Novogene RNA seq protocol