GSM4310178: Sample_Ctrl_KO_1_S1; Homo sapiens; RNA-Seq
For 4sU-seq, HCT116 Ctrl and PPM1G KO cells were seeded into 6-well dishes per biological replicate and grown to 70% confluency. Cells were pulsed for 10 min with DMEM + 500 µM 4sU (Sigma-Aldrich, T4509). At the 9 min mark the DMEM + 4sU media was removed and cells were washed one time with 1X PBS. The PBS was removed, and cells were lysed on-plate using the standard TRizol protocol (ThermoFisher Scientific, 15596026). RNA samples were DNase treated (NEB, M0303) and then extracted with acid:phenol chloroform (ThermoFisher Scientific, AM9720) followed by an additional chloroform extraction. RNA was then precipitated with 1/10 volume of NaCl (5M), 1 µL glycoblue, and 2.5 volumes of 100% EtOH. RNA pellets were washed with 1 mL of 75% EtOH and air dried and then resuspended in RNase-free water. RNA (300 µg) was brought to a final concentration of 0.4 mg/mL with RNase-free water. A separate aqueous master mix (20 mM NaOAC pH 5.2, 1 mM EDTA pH 8.0, 0.1% SDS) was added to the diluted RNA mix followed by the addition of 0.2 mg/mL Biotin-HPDP (ThermoFisher Scientific 21341) in dimethylformamide. The biotinylation reaction was incubated at room temperature for 2 hrs followed by acid:phenol chloroform extraction. Biotinylated RNA pellets were resuspended in 500 µL RNA Prurification Beads (RPB) buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 300 mM NaCl). Dyna MyOne Strepavidin T1 beads (200 uL/sample; ThermoFisher Scientific, 65601) were blocked at room temperature in beads wash buffer + 1% polyvinylpyrrolidone for 10 min. After blocking, beads were washed 1X with 1 mL beads wash buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 50 mM NaCl) and then resuspended in RPB buffer. Biotinylated, 4sU RNA was denatured at 65°C for 5 min and then placed on ice for 2 min. 200 µL of blocked beads were added to each sample and incubated for 30 min with rotation at room temperature. Beads were washed 5X with 4sU wash buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 1 M NaCl, 0.1% Tween-20). Biotinylated, 4sU RNA was eluted from beads 2 times in 75 µL of 0.1 M DTT for 15 min at room temperature with rotation, giving a final elution volume of 150 µL. 4sU-RNA was further purified using the Zymo RNA Clean and Concentrator Kit (Zymo Reasearch, R1013). RNA concentration was measured using the Qubit RNA high sensitivity assay (ThermoFisher Scientific, Q32852) and quality was determined using the Agilent Tapestation RNA ScreenTape (Agilent, 5067-5576).  For RNA-seq, HCT116 Ctrl and PPM1G KO cells were grown in 6-well dishes and total RNA extracted using the Zymo Quick-RNA Miniprep Kit (R1055) according to manufacturer instructions. For 4sU-seq and RNA-seq, the KAPA Stranded RNA-seq kit with RiboErase (HMR) (KAPA Biosystems, KK8483) was used to prepare libraries according to manufacturer's instructions. Briefly, 200 ng of 4sU RNA and 1 µg of total RNA was used to prepare 4sU-seq and RNA-seq libraries, respectively. Libraries were rRNA depleted followed by DNase treatment. RNA was fragmented and primed followed by 1st strand cDNA synthesis. 2nd strand synthesis was then performed with the incorporation of dUTP. Libraries were then A-tailed, adapter ligated, and amplified. All libraries were quantified using the Qubit 2.0 fluorometer and library size verified using the Agilent Tapestation DNA D1000 ScreenTape (Agilent, 5067-5582). All libraries were submitted to the McDermott Center Sequencing Core at UT Southwestern. 75 bp paired-end reads were generated for all libraries. Stranded Whole Transcriptome RNA-seq