GSM7857095: ROI2_fractionated_RNA_biorep2_techrep1_F2; Homo sapiens; OTHER
gDNA extraction: gDNA was extracted from approximately 3-4 million cells with the Cell and Tissue DNA Isolation Kit (Norgen Biotek Corp, 24700), including RNaseA treatment at 37 ˚C for 15 min, and eluting in 200 µL warm elution buffer.  RNA extraction: Approximately 3-4 million cells were solubilized with 1 mL QIAzol (Qiagen, 79306) and 0.2 mL chloroform in 5PRIME Phase-Lock Gel heavy tubes (QuantaBio, 2302830), according to the manufacturer's recommendations. RNA was precipitated at -20 ˚C following the addition of 2 µL GlycoBlue (Thermo Fisher, AM9515) and 2.5 volumes of cold absolute ethanol. RNA was washed once with cold 70% ethanol then resuspended in 30 µL water. 10 µg total RNA or 15 µL of polysomal RNA (half of 30 µL eluate, volumetric inputs) was treated with DNaseI (NEB, M0303) in a 100 µL reaction at 37 ˚C for 15 min, then re-purified by the RNA Clean and Concentrator Kit (Zymo Research, R1015) and eluted in 20 µL water.  Polysome fractionation and polysomal RNA purification: For COMT library stable cell lines, cells at 80-95% confluency (one 15 cm dish per gradient) were incubated with 100 µg/mL cycloheximide (Sigma Aldrich, C4859) in media for 10 min at 37 ˚C, then resuspended in ice cold PBS with 100 µg/mL cycloheximide (PBS-CHX), collected at 4 ˚C by pipetting, and pelleted at 300 x g for 7 min at 4 ˚C. Pellets were flash frozen in liquid nitrogen, then lysed on ice for 10 min with 300-400 µL lysis buffer (20 mM Tris-HCl pH 7.5; 150 mM NaCl; 5 mM MgCl2, 1 mM fresh DTT, 20 mM ribonucleoside vanadyl complex (Fisher Scientific, 50-812-650), 1X protease inhibitor cocktail EDTA-free (Fisher Scientific, 539196), and 100 μg/ml cycloheximide. Lysates were clarified by centrifugation at 1,300 x g for 10 min at 4 ˚C, and 1/10th of the lysate was saved for total RNA extraction. 20-50% sucrose gradients were made with the BioComp Gradient Maker in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 100 μg/ml cycloheximide using Beckman Coulter UC tubes 9/16 x 3-1/2 (Fisher Scientific, NC9194790), and equilibrating overnight at 4 ˚C. Clarified lysates were loaded onto sucrose gradients, and fractionated by ultracentrifugation at 38,000 rpm for 2.5 h at 4 ˚C in a Beckman Coulter SW-41 Ti rotor. Approximately 500 µL polysome fractions were collected using the Biocomp Piston Gradient Fractionator (v8.04) with a Triax flow cell (BioComp model FC-1) and Gilson Fraction Collector using a piston speed of 0.2 mm/s. RNA was precipitated from raw polysome fractions by addition of sodium acetate to 300 mM pH 5.2, 2 µL Glycoblue, and 2x volumes cold ethanol, stored overnight at -20 ˚C. RNA was resuspended in 50-80 µL ultrapure water, and these raw fractions were pooled into metafractions and extracted by phenol-chloroform (5:1) and ethanol precipitation overnight at -20 ˚C. After a single 70% ethanol wash, polysomal RNA was resuspended in 30 µL ultrapure water. Lastly, DNaseI treatment and a column purification was performed prior to library preparation.  Ribosome profiling: Monosomes were pelleted through a sucrose cushion (1 M sucrose, 20 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1x protease inhibitor cocktail EDTA-free) by spinning at 38,000 rpm at 4 ˚C in a SW-41 Ti rotor (Beckman Coulter). Monosome pellets were resuspended in 400 µL lysis buffer and rocked overnight. RNA was extracted with 1.2 mL phenol/chloroform/isoamyl alcohol and precipitated with 300 mM sodium acetate pH 5.2, 1.5 µL Glycoblue, and 2.5 volumes of cold absolute ethanol overnight at -20 ˚C. RNA was pelleted at 21,000 x g for 1 h, washed once with cold 70% ethanol, and resuspended with 30 µL water. 15 µL of RNA was run on a 15% TBE-urea polyacrylamide gel at 150 V for 1.5 h. The gel was stained with 1x SYBR Gold and footprints between 21 nt and 34 nt were cut and isolated by crush and soak in RNA extraction buffer (300 mM sodium acetate pH 5.5, 1 mM EDTA, 0.25% v/v SDS) with overnight rotation. The eluate was passed through a Spin X Centrifuge Tube Filter with a cellulose acetate membrane (Thermo Fisher Scientific, 07-200-385), and the RNA was precipitated with 300 mM sodium acetate pH 5.2, 1.5 µL Glycoblue, and 2.5 volumes of cold absolute ethanol overnight at -20 ˚C. RNA was pelleted at 21,000 x g for 1 h, washed once with cold 70% ethanol, and resuspended in 12 µL water. cDNA synthesis: 10 µL DNaseI-treated total RNA (<10 µg) or DNaseI-treated polysomal RNA (200 ng - 5 µg) was denatured at 65 ˚C for 5 min followed by RT primer annealing at 4 ˚C for 2 min, using 2 pmol pDEST_HC_Rec_Bxb_v2_R primer specific for the landing pad (see Supplementary Table 4) and 2.5 mM random hexamers. Primed total RNA was included in 20 µL SuperScript IV cDNA synthesis reaction (Thermo Fisher, 18090010) with SUPERase-In RNase inhibitor (Thermo Fisher, AM2696), and first-strand cDNA was synthesized by incubating at 23 ˚C for 10 min, 55 ˚C for 1 h, followed by RT inactivation at 80 ˚C for 10 min. RNA was digested with addition of 5 U RNaseH (NEB, M0297) to the first strand cDNA synthesis reaction and incubation at 37 ˚C for 20 min.  Landing pad amplification (PCR1): Nucleic acids (gDNA, 1st strand cDNA) were amplified with Q5 polymerase (NEB, M0491) in 50 µL PCR reactions for 16 cycles with 500 nM landing-pad-specific primers (pDEST_HC_Rec_Bxb_v2_F/R) flanking the entire COMT insert (~1.7 kb). For negative control (plasmid) samples, 10 ng was used as template. For total RNA and polysome RNA samples, 15 µL of unpurified 1st strand cDNA synthesis reaction was used as template. For total gDNA samples, 4 µg template was used in each of three PCR reactions. For flow-sorted gDNA samples, either 1.75 µg template was used in three PCR reactions or 2.5 µg was input into two reactions, with 14 cycles instead of 16. The cycling parameters were: initial denaturation at 98 ˚C for 30 s; 3-step cycling with denaturation at 98 ˚C for 10 s, anneal at 65 ˚C for 30 s, extension at 72 ˚C for 1 min; final extension at 72 ˚C for 2 min. Products from total RNA and polysome RNA samples were purified with the QIAquick PCR Purification Kit (Qiagen, 28106) and eluted in 30 µL elution buffer. Products for total gDNA samples were pooled, resolved on a 0.8% agarose/TAE gel, stained with 1x SYBR Gold, and extracted using the Nucleospin Gel and PCR Cleanup Kit (Takara, 740609) or Zymoclean Gel DNA Recovery Kit (Zymo Research, D4007) with 20 µL 70 ˚C elution buffer. At this point, PCR1 products for gDNA samples cannot typically be seen in the gel, and the expected band size is cut using comparison to a high-molecular weight ladder.  Coding sequence amplification (PCR2): Purified PCR1 products (landing pad insert) were amplified for each of COMT target regions in a 50 µL NEB Q5 reaction (NEB, M0491) for 8 cycles, following the same cycling parameters as for PCR1 and 500 nM tailed primers (see Supplementary Table 4). 15 µL PCR1 product was used as template (50% of eluate for total RNA and polysome RNA samples; 75% of eluate for gel-extracted gDNA samples). Products were purified with the Nucleospin Gel and PCR Cleanup Kit (Takara, 740609) with 1:5 buffer NTI dilution and 25 µL 70 ˚C elution buffer, or with the QIAquick PCR Purification Kit (Qiagen, 28106) with 30 µL elution buffer.  Illumina adapter addition (PCR3): A final Q5 PCR reaction was carried out for 8 cycles with the same formulation as PCR2 but using NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 (NEB, E7600S) according to manufacturer's recommendations (65 ˚C annealing). 25 µL/30 µL eluate was used as template for total gDNA, total RNA, and polysome RNA samples. For flow cytometry gDNA samples, 15 µL/25 µL eluate was used as template. PCR3 products (final library) were purified with the Nucleospin Gel and PCR Cleanup kit (Takara, 740609) and eluted in 25 µL 70 ˚C buffer.  Ribosome profiling: 25 ng of RNA in 8 µL water was input to the D-Plex Small RNA kit (Diagenode, C0503000) and libraries were prepared according to the manufacturer recommendations with the exception that 9 cycles of PCR were carried out. The library was purified with 1.25x AMPure beads (Beckman Coulter, A63880) and quantified by the High Sensitivity DNA Kit (Agilent, 5067-4626).  Quantification and size-selection of final libraries: Final libraries were quantified using the KAPA Library Quantification Kit for Illumina (Roche, KK4873) according to the manufacturer's recommendations, using 1:10,000 dilution of libraries and size-correction with an average fragment size of 300 nt (region 1) and 285 nt (region 2). qPCR was performed on the Applied Biosystems ViiA 7 instrument and values were used to pool individual libraries at equimolar ratios to target ~5 M read pairs per library. Final library pools were size-selected use PAGE purification on a 15% TBE gel followed by a crush-and-soak method (Sambrook and Russell, 2006), or with BluePippin 2% gel (Sage Science, BDF2010) to select fragments from 250-340 bp. Following size-selection, final pools were quantified by High Sensitivity DNA Kit (Agilent, 5067-4626) prior to sequencing. A targeted library preparation method using three sequential PCRs was used (see library construction protocol).