title: GSM5365578: SRSF1 pre-pulldown input Rep2; Homo sapiens; OTHER
library_construction_protocol: After the second wash with 2X quench buffer in the APEX labeling protocol, the cells were harvested by scraping them down in 2X quench buffer including 4 uL Ribolock RNase inhibitor (Thermo Fisher).  The cells were centrifuged for 5 minutes at 200xg at 4 oC.  Next, after aspirating the quench buffer, cells were lysed and processed using the RNeasy plus mini kit (Qiagen) following the manufacture protocol with minor modifications, including adding ß-mercaptoethanol to the RLT lysis buffer, washing with the RWT Buffer (Qiagen #1067933) instead of RW1 buffer, and application of the on-column DNaseI digestion (Qiagen #79256). The RNA was eluted in RNase-free water and quantified using Nanodrop. Same amounts of RNA were aliquoted before the streptavidin pull down of the RNA for downstream applications.  To enrich for biotinylated RNAs, Dynabeads™ MyOne™ Streptavidin C1 magnetic beads (Thermo Fisher) were used with 10 μl of beads per 25 μg of RNA. The beads were washed 3 times in B&W buffer (5 mM Tris-HCl, pH = 7.5, 0.5 mM EDTA, 1 M NaCl, 0.1% TWEEN 20 (Sigma Aldrich)), followed by 2 times in Solution A (0.1 M NaOH and 0.05 M NaCl), and 1 time in Solution B (0.1 M NaCl). Beads were then incubated with purified RNA (25-50ug) in 0.05M NaCl at 4oC for 2 hours with gentle agitations. Following the incubation step, beads were washed 3 times with B&W buffer, followed by the elution of RNA from the beads by adding the TRI Reagent (Sigma-Aldrich T9424) on the beads and extracting according to the manufacturer's protocol. The libraries were prepared by using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® with ribosome-depleted and polyA+ selected RNA