title: HNRNPU RNA targets during neural differentiation using fRIP-Seq
library_construction_protocol: Cells were washed with DPBS, dissociated with TrypLE Express (D0) or Accutase and TrypLE Select and incubated with Neural Isolation Enzyme (D28), and resuspended in Wash medium (DMEM/F12+GlutaMAX, 5% FBS, 10 U/mL penicillin/streptomycin). Cells were gently resuspended and spun at 400 × g for 3 min and resuspended in Wash medium or 1 mL of PBS, supplemented with 0.15 % Triton-X and 30 μL RNase A (Sigma, R4642) to degrade all RNA as described earlier(31). Cells were counted, and wash media was added to 5×106 cells/mL concentration. Cells were crosslinked with 0.1% formaldehyde for 10 min with rotation at room temperature, the reaction was stopped with 125 mM glycine for 5 min with rotation at room temperature, and washed twice in cold DPBS with 1x Halt protease inhibitor cocktail (PIC, Thermo Scientific). Cell pellets were stored at −80°C.  Cell lysis and protein immunoprecipitation were performed following an existing protocol (32) with modifications. Shortly, cell pellets were lysed in polysome lysis buffer (PLB, 100 mM KCL, 5 mM MgCl2, 10 mM HEPES (pH 7.0), 0.5% IGEPAL, 1 mM DTT, 100 units/mL RNaseOUT, 1xPIC). The cell lysate was incubated on ice for 10 min before centrifugation at 10, 000 × g at 4°C. The lysate was transferred to a fresh microfuge tube and pre-cleared by incubating with Dynabeads Protein G (Life Technologies) with 25 μL of beads per sample for 30 min at 4°C with slow rotation. For fRIP-Seq, 5% of the sample was removed for input and stored at −80°C. Protein concentrations were measured using Pierce BSA protein assay kit (Thermo Scientific) and an equal protein amount of lysate was divided into two microfuge tubes per replicate, 1.5 and 1 mg for fRIP-Seq and IP-MS, respectively and NT-2 buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 0.05% IGEPAL, 1 mM DTT, 400 units/mL RNaseOUT, 16.5 mM EDTA) was added to final volume 1 mL. Either the HNRNPU antibody (ab20666, Abcam) or rabbit IgG (12-370, Sigma-Aldrich) was added at a concentration of 7 μg per 1 mg of total protein, and the reactions were incubated for 2 h at 4°C with rotation. Dynabeads protein G was prepared according to the manufacturer's instructions, and 50 µL per 1 mg of total protein was added to fRIP-Seq and IP-MS tubes, respectively. The incubation was prolonged for 1 h. The beads were washed thrice with 1 mL ice-cold NT-2 buffer, rotating for 10 min at 4°C. After a final wash, the supernatant was removed, and beads were stored at −80°C. fRIP-Seq was performed on samples from both CTRLMale and CTRLFemale cell lines at D0 and from CTRLMale at D28. Beads and input samples were subjected to reverse crosslinking. The beads were resuspended to 56 µL Ultra Pure water and 33 µL of reverse crosslinking buffer (PBS--, 6% N-lauroyl sarcosine, 30 mM EDTA, 7.5 mM DTT), 10 mL of Proteinase K and 1 mL of RNaseOUT were added. After incubation (1 h at 42°C then 1 h at 55°C), samples were transferred to 500 μL TRIzol for RNA extraction. Samples in TRIzol reagent (Invitrogen) were phase-separated using chloroform and isopropanol. The aqueous phase was transferred to RNA extraction columns, and the final extraction was carried out with ReliaPrep RNA Cell Miniprep kit (Promega Z6012), including DNA digestion step. An equal amount of RNA, 18 ng, was used for library preparation with Illumina Stranded Total RNA Prep with Ribo-Zero Plus