title: GSM8251650: FLASHseq, CFAP20tagged, rep2; Homo sapiens; OTHER
library_construction_protocol: Cells on 15 cm dishes were washed with 6mL ice-cold PBS and UV-crosslinked with 0.199 mJ/cm2 UV-C light, after which cells were pelleted, snap-frozen in liquid nitrogen and stored at -80˚C until use. Pellets are resuspended in 600µL 1xNLB with protease inhibitors, briefly sonicated with a Bioruptor water bath sonicator (30s ON, 30s OFF, 5 cycles at 4˚C). Lysates were then centrifuged at 20k rcf for 10 minutes at 4˚C to remove insoluble material. Supernatant is transferred to a fresh tube with 25µL MyONE C1 streptavidin beads (Thermo), and incubated in the cold-room with end-to-end rotation for 60 minutes. Beads are washed once with high salt buffer, once with non-denaturing buffer and treated with 0.02U/µL RNaseI (Thermo) in 100µL non-denaturing buffer for 3 minutes at 37˚C, and immediately placed on ice to stop the reaction. Beads are then washed once with high salt buffer, and once with non-denaturing buffer. RNA-ends were repaired with T4 PNK, after which barcoded s-oligos were ligated with T4 RNA Ligase 1 for 90 minutes at 25˚C. The 3'-phosphate at the 3'-end of s-oligos were removed with rSAP, beads are then washed once with lithium dodecyl sulfate-buffer, once with protein lysis buffer, once with high salt buffer, and finally with non-denaturing buffer. The RNA was released with proteinase K treatment, and purified using Oligo Clean and Concentrator columns (Zymo). Reverse transcription was carried out with SuperScript III, samples were then treated with RNaseH (NEB) in order to phosphorylate the 5'-end of the cDNA molecule. After a final round of purification with Oligo Clean and Concentrator columns (Zymo), the cDNA was circularised with CircLigaseII (Lucigen) and amplified with Q5 polymerase (NEB). PCR products were purified with SPRI beads, QCed with Bioanalyzer and subjected to high-throughput sequencing. FLASH-seq (Ilik et al., 2020)