title: GSM8967694: HEK293T Pro-seq DRB 15 minutes Rep1 with Spike; Homo sapiens; OTHER
library_name: GSM8967694
library_construction_protocol: PRO-seq:Cell cultures were collected in 50 mL Falcon tubes and centrifuged in a fixed-angle rotor centrifuge at 600 x g, 4 °C for 5 minutes. The supernatant was removed, and ice-cold PBS (10 mL) was used to wash the pellet (wash repeated 3 times as above). The pellet was then resuspended in 10 mL ice-cold Lysis Buffer. The samples were then incubated for 10 minutes on ice. The samples were centrifuged in a fixed-angle rotor centrifuge at 1000 × g for 10 minutes at 4 °C in 50 mL Falcon tubes. The resulting supernatant was poured off, and the pellet was resuspended with 1 mL lysis buffer, using a wide-mouth P1000 pipette tip. The volume of the sample was brought to 10 mL with lysis buffer and centrifuged at 1000 x g, 4 °C, 5 minutes (wash repeated as above twice). Nuclei were then resuspended in 1 mL freezing buffer (50 mM Tris-HCl pH 8.3, 5 mM MgCl2, 40% Glycerol, 0.1 mM EDTA pH 8.0, brought to volume with 0.1% DEPC-treated DI-water) and transferred to a 1.7 mL Eppendorf tube. Nuclei were centrifuged at 1000 x g, 4 °C, 5 minutes, and the supernatant was removed by pipetting. The pellet was resuspended with 500 μL freezing buffer. The nuclei were again pelleted at 1000 x g, 4 °C, 5 minutes. The supernatant was removed, and the nuclei were resuspended in 110 μL freezing buffer and stored at -80 °C until library preparation. PRO-seq libraries were generated using the qPRO protocol established by Judd et al., 2020. PRO-seq libraries were generated using the qPRO protocol established by Judd et al., 2020. A detailed step-by-step protocol is available at dx.doi.org/10.17504/protocols.io.57dg9i6. In short, ice-cold isolated nuclei were added to 37 °C 100 µl reaction buffer. The reaction was allowed to proceed for 10 minutes at 37 °C. RNA was extracted twice with Trizol, washed once with chloroform, and precipitated with 2.5 volumes of ice-cold ethanol and 1 µl GlycoBlue. The pellet was washed in 75% ethanol before resuspending in 30 µl of DEPC-treated water. The resulting RNA was denatured at 65 °C for 30 seconds and snap-cooled on ice. RNA was then fragmented by base hydrolysis in 0.2 N NaOH on ice for 10–12 min and neutralized by adding a 1× volume of 1 M Tris-HCl pH 6.8, and passed through a P-30 buffer exchange column. The 3' adaptor (/5Phos/GAUCGUCGGACUGUAGAACUCUGAAC/3InvdT/) was ligated with T4 RNA Ligase 1. Biotin-labeled RNA was enriched with streptavidin beads, treated with T4 PNK and RppH for 5' end repair, and ligated to a reverse 5' RNA adaptor (5' UGGAAUUCUCGGGUGCCAAGG). RNA was extracted from beads, and reverse transcribed using SuperScript III. The product was amplified using Phusion DNA polymerase and TruSeq Small RNA Library Preparation Kit barcoded primers. Then the library was size selected with 0.9X AMPure XP beads before being sequenced.