GSM5899107: Ribo-Seq H1299-EV5FUR-2; Homo sapiens; RNA-Seq
RPFs were then extracted using 3V (660 µl) Trizol LS, followed by RNA purification using the Zymo Direct-zol RNA micro prep kit with modifications to the manufacturer's instructions: Columns were spun dry for 2 min at 12,000 g and isolated RPFs were eluted in 20 µl nuclease-free water. Ribosome profiling was performed as described in (McGlincy and Ingolia, 2017; van Heesch et al., 2019), with several modifications. Snap-frozen cells were scraped off in 1 ml of ice-cold lysis buffer (20 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 0.1% IGEPAL CA-630, 1 mM DTT, 10 U/mL RNase-free DNase 1, 100 µg/mL cycloheximide) and transferred to Eppendorf tubes for lysis on ice for 10 min. Lysates were clarified through centrifugation at 20,000 g at 4°C for 10 min. Total RNA content of sample lysates was estimated using the Qubit™ RNA broad range (BR) Assay Kit on a Invitrogen™ Qubit™ 4 fluorometer. Lysates were then digested per 200 µl aliquots with RNAse I for generation of ribosome-protected fragments (RPFs), using 20 units of RNAse I per 20 µg of RNA measured in lysate. After 45 min of digestion at 23°C while shaking at 400 RPM on a thermomixer, the digestion reaction was stopped by adding 10 µl (10U) of SUPERase*In RNAse inhibitor and placing the samples on ice. Digested lysates were then transferred to Microspin S-400 HR sephacryl columns equilibrated with 3 ml of cold polysome buffer (20 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM MgCl2) each and centrifuged at 600 g at room temperature for 2 min using an Eppendorf 5425 R microcentrifuge. 20 µl 10% SDS were then added to digested lysates and RPFs were then extracted using 3V (660 µl) Trizol LS, followed by RNA purification using the Zymo Direct-zol RNA micro prep kit with modifications to the manufacturer's instructions: Columns were spun dry for 2 min at 12,000 g and isolated RPFs were eluted in 20 µl nuclease-free water. rRNA was depleted using RiboPOOL technology with modifications to the manufacturer's instructions: 200 pmol of RiboPOOL and 100 µl of beads were used per sample. RNA was purified using the Zymo RNA Clean and Concentrator-5 kit with modifications to the manufacturer's instructions as mentioned above. Purified RNA was then size-selected through denaturing PAGE using 15% TBE-Urea gels. RNA fragments corresponding to 26-34 nucleotides were excised and recovered from gel slices by rocking at 37°C at 700 RPM on a thermomixer. RNA solutions were transferred to Costar Spin-X filter tubes and filtered through centrifugation at 5000 RPM for 6 min using an Eppendorf 5425 R microcentrifuge. 2 µl of GlycoBlue and 700 µl of isopropanol were then added per aliquot and RNA was left to precipitate overnight at -80°C. Following precipitation, RNA fragments were pelleted by centrifugation at 21,000 g at 4°C for 45 min. Pellets were washed once with 800 µl of ice-cold ethanol and centrifuged at 21,000 g at 4°C for 15 min. RNA pellets were then air-dried for 3-4 min and dissolved in 60.75 µl nuclease-free water on ice. RNA fragments were dephosphorylated using 30 U of T4 PNK for 1 h at 37°C and purified using Zymo RNA Clean and Concentrator-5 kit, where isolated RNA fragments were eluted in 9.5 µl nuclease-free water. Purified fragments were then ligated to a pre-adenylated 3'oligonucleotide linker using 100 U of T4 RNA ligase 2 Deletion Mutant and 5 U T4 RNA ligase 1 at 23°C for 3 h. Leftover linker was removed using 5'Deadenylase and Rec J Exonuclease. Linker-ligated RNA fragments were reverse-transcribed into cDNA using EpiScript reverse transcriptase. cDNA was treated with Exonuclease I for 30 min at 37°C, followed by 15 min at 80°C with a reduction to 4°C: and further treated with 5 U of RNAse I and 2.5 U of Hybridase Thermostable RNase H at 55°C for 5 min followed by an incubation step at 4°C to stop the reaction. Treated cDNA was purified using the Zymo Oligo Clean and Concentrator Kit with modifications to the manufacturer's instructions: Columns were spun dry for 2 min at 12,000 g and isolated RPFs were eluted in 9.5 µl nuclease-free water. cDNA containing ligated linkers was size-selected through denaturing PAGE using 10% TBE-Urea gels. cDNA fragments corresponding to 70-80 nucleotides were excised and precipitated with ammonium acetate and SDS followed by overnight precipitation with isopropanol as described above. Size-selected cDNA was circularised for 3 h at 60°C using 100 U of circLigase I followed by heat inactivation for 10 min at 80°C and amplified using Phusion high-fidelity polymerase (New England Biolabs) with reverse primers containing unique barcode sequences for 10 cycles of: 30 sec at 98°C, 15 sec at 94°C, 5 sec at 55°C, 10 sec at 65°C. Following amplification, 5 µl of 3 M NaCl, 1 ml of ethanol and 2 µl of GlycoBlue were added per aliquot cDNA left to precipitate overnight at -80°C. Amplified cDNA libraries were size-selected using 7% non-denaturing TBE-Urea gels. cDNA libraries corresponding to 150 nucleotides were excised and recovered from gel slices by rocking at 37°C at 700 RPM on a thermomixer. cDNA solutions were then transferred to Costar Spin-X filter tubes and filtered through centrifugation at 5000RPM for 6 min using an Eppendorf 5425 R microcentrifuge. cDNA libraries were purified using the Zymo DNA Clean and Concentrator-5 kit with modifications to the manufacturer's instructions: Columns were spun dry for 2 min at 12,000 g and isolated RPFs were eluted in 13 µl nuclease-free water. cDNA libraries were quantified using Qubit™ DNA high sensitivity (HS) Assay Kit according to the manufacturer's instructions on an Invitrogen™ Qubit™ 4 fluorometer and Bioanalyzer 2100 using the High Sensitivity DNA kit and pooled in equimolar ratios. Sequencing was performed on a NextSeq2000 following standard Illumina protocols.