Tip sonicator

ChIP was performed as described previously [44 (link)], with the following modifications: chromatin was sheared by using a tip sonicator (Fisher Scientific) for 25 rounds of 20 s pulses with 20 Amplitude and 40 s off. Chromatin was diluted in ChIP dilution buffer (0.01% [w/v] SDS, 1.1% [v/v] Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl). ChIP was performed overnight at 4 °C using 5 μg RNAP II antibody (8WG16, Millipore), FUS (J2516, Santa Cruz), Ser2 RNAPII (5095, Abcam), with Control IgG (Cell Signaling) and rotated overnight at 4 °C. DNA was isolated after crosslink reversal using a QIAGEN PCR clean up kit prior to qPCR (Table S1).

Following plasmid isolation of sequence-verified clones, plasmids were transformed into BL21(DE3) competent E. coli cells (NEB). Glycerol stocks of BL21(DE3) E. coli harboring the desired genes supplemented with kanamycin were kept at -80 °C. Starter cultures (4 ml 2xYT medium, 45 µg/ml kanamycin) were inoculated from these stocks, grown overnight and used to inoculate 1 L of 2xYT medium containing kanamycin (45 µg/ml). The cultures were shaken (200 rpm) at 37 °C to an OD₆₀₀ of 0.8 and induced by the addition of 0.5 mM IPTG and allowed to grow overnight in the shaker incubator. Cells were harvested by centrifugation (5k rcf, 15 min, 4 °C) and resuspended in 25 mL of 100 mM PBS buffer (pH 7.6). E. coli cells were lysed using a tip sonicator (Fisher Scientific) following by centrifugation (18k rcf, 25 min, 4 °C). Supernatants (termed soluble fraction) were subjected to purification.

Electrospinning was performed by adding 20 mL of dimethyl formamide (DMF) (Fine-Chem Limited, industrial Estate, Mumbai, India) solvent into a 3 gm polyvinylidene difluoride (PVDF) to get 15 wt % PVDF concentration. A plastic syringe tipped with a stainless steel needle was filled with 3 mL of the PVDF solution. The positive voltages came from a high voltage supply CZE1000R (Spellman, Hauppauge, NY, USA) to the metal needle, for application of bias values around (25 kV) with constant rate of (1.5 mL/h) using a syringe pump NE1000 (New Era Pump Systems, Suffolk County, NY, USA) with needle-to-collector distance of 10 cm. Random PVDF nanofibers were obtained using a normal metal plate collector covered with aluminum foil which was connected to ground. For comparison, Aligned PVDF fiber was fabricated by 2-metal bars of 1.5 cm length, as shown in Figure 1. The addition of MWCNTs with different concentration (0.1 and 0.3 wt %) was introduced by dispersing the CNTs into PVDF solution with the aid of tip sonicator (Fisher Scientific, Hampton, NH, USA) for 15 min in an ice bath to diminish the effect of heat on the CNT structure.