GSM5618624: Ezra_seq_HEK293_Starvation_rep2; Homo sapiens; OTHER
A total of 4 plates (10-cm) HEK293-Kb cells grown to 80% confluency were changed with fresh medium to remove the dead cells 2 h before harvesting.Cells were washed by cold PBS and lysed in the polysome lysis buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2, 100 μg/mL cycloheximide with 2 % Triton X-100). The lysates were cleared by centrifugation at 14,000 rpm for 10 min at 4 °C. 15-45 % (wt/vol) sucrose density gradients were freshly prepared in a SW41 ultracentrifuge tube using a Gradient Master. 500 μL of cytosolic extracts were loaded onto sucrose gradients followed by ultracentrifugation for 2 h 30 min at 32,000 rpm 4 °C in a SW41 rotor. Polysome profiles were recorded at A254 using the Brandel Gradient Fractionation System and an ISCO UA-6 UV/Vis detector. An aliquot of ribosome fractions representing monosome or polysome were collected followed by digestion with E. coli RNase I (750 U per 100 A260 units) by incubation at 4 °C for 1 h. Total RNA was extracted using Trizol LS reagent. Purified RNAs were used for cDNA library construction. The ribosome-protected mRNA fragments were separated on a 15% polyacrylamide TBE-urea gel and visualized using SYBR Gold. Selected regions in the gel corresponding to 25–35 nt were excised. RNA fragments were dissolved by soaking overnight in 400 μL RNA elution buffer (300mM NaOAc, pH 5.2, 1mM EDTA, 0.1 U mL−1 SUPERase_In). The gel debris was removed using a Spin-X column, followed by ethanol precipitation. Purified RNA fragments were resuspended in nuclease-free water and quantified using Qubit 2.0 Fluorometer. A fraction of RNAs (10~200 ng) were used for cDNA library construction. In brief, 10 μL RNAs were mixed thoroughly with 1 μL T4 PNK (10 U), 1 μL purified TS2126 and 1 μL PAP1 enzyme in 7 μL Ezra buffer). The mixture was incubated at 37°C for 30 min followed by 65°C for 20 min. After ethanol precipitation at -20°C for 30 min, the dissolved RNA pellet was mixed with 0.5 μM 5' end adaptor followed by ligation for 90 min at 22 °C in a 10 μL reaction mixture (1 × T4 Rnl2 reaction buffer, 10 U SUPERase_In, 15% PEG8000 and 20 U T4 RNA ligase 2 truncated KQ). The ligated RNA sample was mixed with 1 μl 0.5 mM dNTP and 1 μl 0.5 μM RT primer and incubated at 65 °C for 3 min, followed by incubation on ice for 1 min. The reaction mix was then added with 9 μl cDNA synthesis mix (5 × first strand buffer, 0.1 M DTT and 750 μg/μl m-MLV-mut5) followed by incubation at 50°C for 30 min. The synthesized cDNAs were mixed with 10 μL rRNA depletion oligo mix tagged with biotin (1 μL 25 μM oligo mix and 3 μL 20 × SSC buffer) and heated at 94°C for 30 s followed by slowly cooling down (3 °C/min) to 37 °C. 30 μL pre-washed streptavidin magnetic beads was added to each sample and stay at room temperature for 10 min. After placing on magnet stand for 1 min, the supernatant was transferred to a new tube and precipitated by ethanol. rRNA depleted cDNAs were amplified by PCR using barcoded sequencing primers. The PCR contains 1 × HF buffer, 0.2 mM dNTP, 0.5 μM oligonucleotide primers, and 0.5 U Phusion polymerase. PCR was carried out under the following conditions: 98 °C, 30 s; (98 °C, 5 s; 68 °C, 15 s; 72 °C, 10 s) for 12 cycles; 72 °C, 3 min. PCR products were separated on a 8% polyacrylamide TBE gel. Expected DNA at 180 bp was excised and recovered from DNA gel elution. After quantification by Agilent BioAnalyzer DNA 1000 assay, equal amounts of barcoded samples were pooled into sequencing lanes, which were sequenced using NextSeq 500.