GSM3613475: small RNA-seq of total rep1; Mus musculus; ncRNA-Seq
Total RNA was extracted by TRIZOL reagent. For RIP-seq, N2a cells cultured in a 150 mm plate were washed twice with cold PBS followed by UV irradiation at 400mJ/cm2 under a HL-2000Hybrilinker. Scrapped cells were harvested by centrifugation at 500 g for 5 min at 4°C. Crosslinked cells were lysed in 1 ml lysis buffer (20mM Tris-Hcl pH 7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 0.1% SDS, 0.1% deoxycholate, 100U/ml RNasin Ribonuclease Inhibitors (Promega), 1 u/ml EDTA-free complete protease inhibitor cocktail (Roche)) on ice for 30 min. Lysates were centrifuged at 12,000 g at 4°C for 15 min to remove cell debris. An input of collected supernatant was set aside for subsequent analysis. Each sample was then divided and incubated with 5 μg of either anti-TDP-43 antibody (Proteintech) or rabbit IgG (Millipore) on a rotator overnight at 4°C. Complexes were pulled down by incubation with protein G Dynabeads on a rotator for 2 hours at 4°C. Beads were washed twice with lysis buffer, twice with wash buffer (add 300 mM NaCl), and boiled to reverse UV cross-linking. Co-precipitated RNAs were extracted from beads and used for construction of small RNA library or analysis by quantitative RT-PCR (qRT-PCR) using specific primers.To isolate cellular insoluble aggregates, two 10-cm dish N2a cells were extracted sequentially, as previously described (Guo et al., 2011; Hu et al., 2017). Briefly, cells were harvested, lysed in the moderate RIPA lysis buffer (50mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, EDTA-free complete protease inhibitor cocktail (Roche), 100U/ml RNasin Ribonuclease Inhibitors (Promega)) on ice for 30 min, and centrifuged at 17000 g at 4°C for 30 min. The bottom cell debris (i.e. nuclear, mitochondria and stress granules) were depleted. The resultant supernatant fractions were incubated with 1% Sarkosyl for 30 min at room temperature with agitation, and then the detergent insoluble proteins were obtained by ultracentrifugation of the supernatants at 41600 rpm at 4°C for 30 min and washed twice by PBS buffer. The whole cell lysates and the detergent-insoluble pellets were separately collected, and the isolated protein and RNA were used for Western blotting analysis and small RNA-seq library construction.The small RNA library was constructed using sensitivity-improved method as described (Yang et al., 2016) or modifications. Briefly, isolated total RNA (~1 μg) was mixed with 25 pmol 3′ adapter, 1 × T4 ligase reaction buffer, 33% polyethylene glycol (molecular weight 8000), 40 U RNL2tr K227Q (NEB), and 20 U RiboLock RNase Inhibitor (Thermo Fisher). The samples were incubated at 22°C for 2 hours, then at 16°C for 1 hour, followed by addition of 1 μl of 10 μM RT primer before heat inactivation at 75°C for 5 min. 200 pmol of the 5′ adapter was added for ligation by T4 RNA ligase 1 (NEB) and incubated at 22°C for 1 hour. Linker-ligated RNAs were reverse transcribed with Superscript III (Invitrogen) at 50°C for 30 min, 55°C for 30 min, then at 95°C for 5 min and PCR-amplified with Q5 High-Fidelity 2× Master (NEB). The product was fractionated on a 4% agarose gel and extracted by QIAEX II Gel Extraction Kit (QIAGEN). The recovered DNA was quantified for deep sequencing.