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Cell cycle specific, differentially tagged ribosomal proteins to measure phase specific transcriptomes from asynchronously cycling cells – Scientific Reports

The Flp-In HEK293-T cells stably expressing ccTaggedRP cell line and plasmid are available upon reasonable request to the corresponding author. The CAG-ccTaggedRP plasmid will be deposited into Addgene.

Creation of the ccTaggedRP construct

A DNA construct encoding 3× Flag-Tag, human Cdt1 (amino acid 30 to 120), T2A, 3× HA-Tag, and human Geminin (hGem (amino acids 1 to 110)) and containing 5P BglII and 3P NheI restriction sites was synthesized by IDT DNA, Inc. The hCdt1 and hGem DNA sequences were similar to the sequences used in Fucci2aR22. CMV promoter and multicloning sites were removed from pSF-CAG-Kan (plasmid OGS505, Sigma, Inc) using BglII and NheI and replaced with the sequence containing 3× Flag Tag, hCdt1(30–120), T2A, 3× HA Tag, and hGem(1–110). The DNA sequence corresponding to cDNA of mouse RPL10a was synthesized by IDT DNA, Inc, and modified to add 5P and 3P AvrII or 5P and 3P MluI restriction sites. The add 5P and 3P AvrII or 5P and 3P MluI RPL10a cassettes were subcloned into 3× Flag Tag, hCdt1(30–120), T2A, 3× HA Tag, and hGem(10–110) to create in frame 3× Flag Tag-RPL10a-Cdt1(30–120)-T2A-3× HA Tag-RPL10a-hGem(10–110) in the pSF plasmid. Then, a CAG promoter was subcloned into the 3× Flag Tag-RPL10a-hCdt1(30–120)-T2A-3× HA Tag-RPL10a-hGem(10–110) plasmid to create the CAG-ccTaggedRP construct. We made the Flp-In HEK293-T cells stably expressing ccTaggedRP using the ccTaggedRP DNA construct that subcloned into a plasmid modified to contain a CAG promoter and 3P FRT and Hygromycin sequences corresponding to pcDNA5/FRT (ThermoFisher, Inc.). The Flp-In HEK293-T cells were purchased from Invitrogen, Inc and the Flp-In HEK293-T cells stably expressing ccTaggedRP were made according to the manufacture’s protocol. The DNA sequences of all constructs were validated by Sanger sequencing.

Polysome profiling

Polysome profiling of HEK293T cells was conducted as described previously33. A 100 mg cell pellet was lysed with 500 µL of lysis buffer (20 mM HEPES–KOH pH 7.7, 100 mM KCl, 5 mM MgCl2, 0.5% IGEPAL, 1 mM DTT, 0.5× protease inhibitor cocktail (Promega), 4 units/mL RNase inhibitor (Promega), 100 µg/mL cycloheximide). The lysate was passed through a 23G needle four times and centrifuged at 11,200×g at 4 °C, for 8 min. The clarified lysate was loaded on top of a linear sucrose gradient (10–50% sucrose, 20 mM HEPES–KOH pH 7.7, 100 mM KCl, 10 mM MgCl2, 1 mM DTT). The sample was centrifuged at 230,000×g at 4 °C, for 2.5 h using an SW40 rotor. After centrifugation, a BioComp gradient profiler was used to fractionate the sucrose gradient at 0.2 mL/min into 800 µL fractions. The absorbance at 260 nm was measured during fractionation.

Negative stain EM

For negative stain electron microscopy, a 10 µL sample corresponding to the 80S ribosome peak (fraction 5) was applied directly after fractionation to a glow discharged 400-mesh grid (EMS, CF400-Cu-50) and incubated for 5 min at room temperature. The grid was stained with 2% uranyl acetate twice for 1 min each. Excess fluid was blotted away with Whatman filter paper after each incubation period (Cytiva, 1001-090). Images were collected on an FEI F20 electron microscope operated at 120 kV and equipped with a TVIPS TemCam XF416 camera. Images were taken at a magnification of 62,000×, and a pixel size of 0.17 nm.

Western blotting

For Western Blotting of sucrose gradient fractions, TCA (100% w/v) was added to each fraction at a final concentration of 10% and the samples were incubated for 30 min on ice. The samples were centrifuged at 13,000×g at 4 °C for 15 min. Pellets were washed with ice cold acetone and centrifuged at 13,000 xg for 10 min (2×). The pellets were dried, then resuspended in 100 µL of 1× SDS-PAGE sample buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 6% glycerol, 0.004% bromophenol blue, 1% β-mercaptoethanol). Proteins were resolved on a NuPAGE 4–12% Bis–Tris minigel (Invitrogen, NP0323BOX) and transferred onto a 0.2 µm PVDF membrane (Invitrogen LC2002). The membrane was blocked in 3% BSA in 1× PBST (0.1% Tween-20) and probed with FLAG (Sigma, F1804), HA (Invitrogen, 26183), and RPL10a (Invitrogen, MA5-44710) antibodies diluted in 1% BSA in 1× PBST. Secondary anti-mouse (Invitrogen, A21058) and anti-rabbit (LI-COR, 926-32213) antibodies were used for detection with the LI-COR Odyssey imager.

For Western blotting of cell lysates, HEK293-T cells expressing ccTaggedRP were cultured in DMEM (CAT#:11965-092, ThermoFisher) supplemented with 10% fetal bovine serum (CAT#: 10438026, ThermoFisher) and 500 µg/mL Hygromycin B (CAT#: 10687010, ThermoFisher) until 80% confluency. Cells were washed once with Dulbecco’s phosphate-buffered saline (DPBS) (CAT#: 14040133, ThermoFisher) and lysed in RIPA buffer (CAT#: 89900, ThermoFisher) supplemented with protease inhibitors (Roche, 04693159001). Protein concentration was quantified with the Pierce BCA kit (CAT#: 23225, ThermoFisher) and normalized. 4× loading buffer was added to samples. 25 µg of total protein was run on SDS gels (CAT#: 4561034, Bio-rad) and transferred to PVDF membranes with 45 µm pores (CAT#: 84-895, Prometheus). The membranes were blocked in 5% non-fat milk (Great Value) in Tris-buffered saline with 0.1% Tween 20 (TBST). Membranes were then incubated overnight with primary antibodies against FLAG (1:1000, CAT#: F1804, Invitrogen), HA (1:1000, CAT#: 26183, Invitrogen), or GAPDH (1:1000, CAT#: ab9485, Abcam) in 5% milk solution. Membranes were washed three times with TBST and subsequently incubated in TBST with HRP-conjugated secondary antibodies (CAT#: G-21234, Invitrogen). Membranes were washed twice with TBST and once with TBS. Chemiluminescent reaction was catalyzed by addition of 1:1 1 Ambersham ECL Detection Kit. Membranes were imaged on a Bio-Rad ChemiDoc Touch Imaging System (Bio-Rad, Inc.). Densitometry was quantified using Bio-Rad Image Lab 6.1 (Rio-Rad, Inc.).

Serum starvation of cultured cells

HEK293-T cells expressing ccTaggedRP were cultured in DMEM (CAT#:11965-092, ThermoFisher) supplemented with 10% fetal bovine serum (CAT#: 10438026, ThermoFisher) and 500 µg/mL Hygromycin B (CAT#: 10687010, ThermoFisher) until 80% confluency. Starved samples were then cultured in DMEM supplemented with only fetal bovine serum for the indicated amount of time.

Pharmacologic synchronization of cultured cells

HEK293-T cells expressing ccTaggedRP were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (CAT#:11965-092, ThermoFisher) supplemented with 10% fetal bovine serum (CAT#: 10438026, ThermoFisher) and 500 µg/mL Hygromycin B (CAT#: 10687010, ThermoFisher) until 70% confluency. Cells were then treated with vehicle, 100 μM Lovastatin (CAT#: 400046, Sigma) for 48 h, 20 µg/ml hydroxyurea (CAT#: 400046, Sigma) for 24 h, or 5 µg/ml nocodazole (M1404, Sigma) for 48 h.

Flow cytometry

Cells were incubated with 10 µM EdU for 1.5 h prior to harvest. Following harvest, cells were incubated with fixable LIVE/DEAD stain (CAT#: L34992, ThermoFischer) for 20 min in the dark and then washed with PBS. Following staining, cells were fixed with 4% paraformaldehyde (PFA) for 20 min and then were washed with 1% BSA. Cells were permeabilized and underwent the Click-it reaction according to manufacturer’s instructions (CAT#: C10633, ThermoFischer). Cells were then washed with permeabilization buffer and stained with FxCycle Violet (CAT#: F10347, ThermoFischer) for 1 h in the dark. Data was obtained with a BD LSR Fortessa and analyzed with OMIQ (Dotmatics). Gating strategy is illustrated in Supplemental Fig. 2.

Immunofluorescence of cultured cells

HEK293-T cells expressing ccTaggedRP were cultured in DMEM (CAT#:11965-092, ThermoFisher) supplemented with 10% fetal bovine serum (CAT#: 10438026, ThermoFisher) and 500 µg/mL Hygromycin B (CAT#: 10687010, ThermoFisher) until 80% confluency. Medium was removed, and cells were fixed in 4% PFA. Cells were washed and incubated overnight in 1× PBS. Slides were then incubated at 90 °C in 1× unmasking solution (CAT#: H-3300, Vector Labs). Slides were cooled to room temperature and then washed three times with 1× PBS. Slides were blocked in 1× PBS containing fish skin gelatin oil (FSG) and Donkey Serum (CAT#: S30-100ML, Millipore Sigma) for 1 h at room temperature. Cell slides were incubated in primary antibodies against FLAG (CAT#: PAB29056, Abnova) and RPL10a (CAT#: PA5-62845, Invitrogen) in 1× PBS contacting FSG overnight at 4 °C. Slides were washed once with 1× PBS containing FSG and 0.1% Tween-20 and once with 1× PBS. Cells were then incubated with secondary antibodies against donkey anti-chicken (CAT#: 703-585-155, Jackson ImmunoResearch) and donkey anti-rabbit IgG (CAT#: 711-475-152, Jackson ImmunoResearch) in 1× PBS containing FSG and 0.1% Tween-20. Slides were washed once with 1× PBS containing FSG and 0.1% Tween-20 and once with 1× PBS. Cover slips were then mounted with VECTASHIELD® Antifade Mounting Medium with DAPI (H-1200-10, Vector Laboratories).

Cell culture, lysis, immunoprecipitations, and RNA recovery

HEK293-T cells expressing ccTaggedRP were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (CAT#:11965-092, ThermoFisher) supplemented with 10% fetal bovine serum (CAT#: 10438026, ThermoFisher) and 500 µg/mL Hygromycin B (CAT#: 10687010, ThermoFisher) until 70% confluency. Cells were washed with DPBS (CAT#: 14040133, ThermoFisher) and lysed in Pierce™ IP Lysis Buffer (CAT#: 87787, ThermoFischer) supplemented with 1× protease inhibitor (CAT#: 87785, ThermoFischer) and RNAse Inhibitor (CAT#: AM2682, Invitrogen). Lysed samples were incubated overnight with 5 µg of IP antibodies against FLAG (CAT#: F1804, Invitrogen) or HA (CAT#: 26183, Invitrogen). 0.25 mg of Pierce Protein A/G Magnetic Beads (CAT#: 88802, ThermoFischer) were added, and the resulting mixture was incubated overnight with rocking at 4 °C. Magnetic bead-target complexes were collected with a magnetic stand, and the remaining solution was aspirated. Samples were washed twice with IP lysis buffer and resuspended in RW1 buffer. RNA was then isolated with the RNeasy mini kit (CAT#: 74104, Qiagen) according to the manufacturer’s instructions. Isolated mRNA samples were quantified and submitted to Genewiz for further processing and sequencing.

RNA sequencing analyses

cDNA library preparation with adapter ligation, and sequencing using an Illumina® HiSeq® system were performed by Genewiz, Inc. Reads underwent quality control, trimming, alignment, and quantification according to Genewiz standard workflows. Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. BAM files were generated as a result of this step. Below are the statistics of mapping the reads to the reference genome. Genes with less than ten reads in at least three samples (the minimum sample size for a group) were excluded for subsequent analysis. Differential expression analysis was performed using R and the DEseq2 package. Functionalization was performed using ClusterProfiler package in R and GSEA software.

scRNAseq analysis

A scRNAseq dataset of cultured HEK293-T cells was sourced from 10× Genomics (10× Genomics). Sequencing yielded 737,280 cells with an average of 66.58 reads per cell and 19.45 genes per cell. Poor quality cells were removed, using the following inclusion criteria: cells with greater than 2000 genes, greater than 7500 reads, and less than 7.5% mitochondrial DNA. Cells were assigned a “G2/M” or “G1/S” identity using the CellCycleScoring algorithm in Seurat and previously published data sets (supplemental Fig. 7)34,35. Cells unable to be assigned confidently to either identity were discarded before downstream analysis. After filtering, 2447 cells remained with an average of 15,651.61 reads per cell and 3485.21 genes per cell. Data was normalized and scaled with the Seurat package in R. UMAP dimensionality reduction was performed with 11 principal components and a resolution of 0.5. Differential expression analysis based on the cell cycle cluster was performed using the Seurat package in R. Functionalization was performed using clusterProfiler package in R.

Quantification and statistical analysis

Shapiro Wilk test was performed on continuous variables to evaluate the data distribution. Normally-distributed data and non-normally distributed data were then analyzed for statistical significance by Student t test and Mann–Whitney U test, respectively. Statistical significance for RNAseq and scRNAseq data was determined through Benjamini–Hochberg procedure and reported as the adjusted p-value. All data is presented as mean ± standard deviation.

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