Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Schematic of library generation by OTTR or ligation-based protocols from a single pool of RNase I derived RPFs (green) from a sucrose cushion. The pool of RPFs were split unevenly after T4 PNK treatment with only 1:10th of the RPFs used in OTTR. In OTTR, each step before cDNA size selection occurs in 4 hours in a single tube. First, input RPF RNA was labeled by either ddA or ddG on the 3′ end before unincorporated ddRTPs were inactivated by rSAP. Lastly, two ordered jumps, the first initiated from the +1Y DNA/RNA primer duplex and the second initiated from a non-templated dG addition to the RPF cDNA to jump to the 3’C adapter template, yields a cDNA molecule with a 5’ and 3’ adapter flanking the complement of the RPF input. In ligation-based, the 3’ adapter is first adenylated on its 5’ end. THen, 3’ adapter ligation is carried out, followed by gel-based size selection and overnight elution. THe next day, material is precipitated and primer hybridization for reverse transcription occurs. Following reverse transcription, cDNA is purified by gel-base size-selection. After elution, cDNA is circularized. In these illustrations green/light green denoted the RPF sequence, orange/light orange denoted the R1 adapter sequence, blue/light blue denoted the R2 adapter sequence, gray/dark gray denoted the unique molecular identifier sequence, brown/light brown denoted the barcode sequence, red octagon denoted polymerase blocking groups, magenta triangle denoted a 3’ddR, and a magenta square denoted the dG non-templated addition. b, Comparison of gene-level ribosome occupancy estimates from libraries generated in (a). Read counts are for RPFs aligned to verified CDSs excluding those RPFs that are aligned to the first 15 and last 10 codons. Read counts for each gene were normalized by DESeq2. c, Comparison of mean codon-level occupancy estimates from libraries generated in (a). Aligned RPFs were assigned to an A-site codon and counted. These counts were then rescaled by the mean codon count for the gene, excluding those RPFs that are aligned to the first 15 and last 10 codons, and averaged across the translatome. d-e, Metagene averages around the start (left) and stop (right) codons for either (d) OTTR or (e) ligation-based libraries. Aligned RPFs for each CDS were first rescaled by the mean codon count for the gene, excluding those RPFs that are aligned to the first 15 and last 10 codons, and then averaged across the translatome. Footprints were tabulated according to either the 5′ aligned position alone (shown at top as a black line), or both 5′ aligned position and read length (shown at bottom as a matrix of distinct RPF lengths and positions). f, Per-codon contributions to iχnos machine learning models of RPF occupancy profiles. A model based on a widow of 13 codons (−7 to +5) around the A-site was compared with thirteen additional models, each omitting one codon from the model. The contribution of a codon position to RPF occupancy profile was inferred from the change in Pearson’s correlation coefficient between the predicted ribosome occupancy versus actual ribosome occupancy changed when the codon was omitted (Y-axis).

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Ligation, Derivative Assay, Selection, Labeling, Hybridization, Reverse Transcription, Purification, Sequencing, Blocking Assay, Comparison, Generated

Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Size-selection of P1 nuclease RPF cDNA from OTTR by direct imaging of Cy5, the dye covalently linked to the 5′ end of the +1dY DNA/RNA adapter duplex primer (see Fig. 1a). The 30 nt and 40 nt RNA oligonucleotides used for RPF size selection (not shown) were also used in OTTR reactions parallel to those using input RPFs, to generate cDNA size-selection markers. Bromophenol blue formamide loading dye was used to resuspend OTTR cDNA for size selection to avoid xylene cyanol interference during Cy5 imagining. A 0.6X TBE 8% urea-PAGE was chosen for cDNA size selection since xylene cyanol and the no-insert OTTR adapter-dimer cDNA (~75 nt) co-migrate. For these reasons, xylene cyanol was included only in the peripheral lanes. Horizontal black lines indicate the boundaries for cDNA gel slice excision to remove adapter-dimer from desired cDNA library. All lanes are from the same gel. P1 RPF were either from sucrose cushion purified human 293T or sucrose cushion purified S288C yeast material after nuclease digestion. A 30 nt and 40 nt template control OTTR reaction was used to synthesize OTTR cDNA to enable cDNA size selection equivalent to RNA size selection. b, Read length distribution of yeast RNase I (blue) and P1 nuclease (red) RPFs from the CDS, excluding those sucrose cushion purified RPFs that are aligned to the first 15 and last 10 codons, as in Extended Data Fig. 1b. Counts were represented in RPM and averaged across replicates. c, Read length distribution of human RNase I (blue) and P1 nuclease (red) RPFs from the CDS, excluding those sucrose cushion purified RPFs that are aligned to the first 15 and last 10 codons, as in Extended Data Fig. 1b. Counts were represented in RPM and averaged across replicates d, Fraction of sucrose cushion purified RNase I RPF cDNA library sequencing reads mapped to each transcript class for yeast libraries generated by P1 nuclease or RNase I digestion. e, Fraction of sucrose cushion purified RNase I RPF cDNA library sequencing reads mapped to each transcript class for human libraries generated by P1 nuclease or RNase I digestion. f, Average per-base read coverage of cytosolic 18S and 25S or 28S rRNA from yeast (left) or human (right) ribosome profiles with P1 nuclease (red) or RNase I (blue). Coverage was represented in reads per million total reads, including reads mapping to rRNA, tRNA, ncRNA, mRNA, and other genomic loci) to emphasize relative proportion from the entire library. Material was purified from a sucrose cushion. g, As in (f) but for 5.8S and 5S rRNA coverage. h, As in (f) for mitochondrial rRNA coverage.

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Selection, Imaging, cDNA Library Assay, Purification, Control, Sequencing, Generated

Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Fraction of RNase I RPF cDNA library sequencing reads mapped to each transcript class. Library generation artifacts included sequences that were adapter-only, shorter than 15 bases, or unmapped. b, Read length distribution of OTTR (blue) and ligation-based (red) RNase I RPFs from the CDS, excluding those RPFs that are aligned to the first 15 and last 10 codons. Counts were represented in RPM and averaged across replicates c, For each read length from 26 to 29 nt, the fraction of RPF alignments with mismatches at the 5′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference. d, For each read length from 26 to 29 nt, the fraction of RPF alignments with an adenosine (A) at the 3′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference. e, For each read length from 26 to 29 nt, the fraction of RPF alignments with a thymine (T) at the 3′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference.

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Comparison, Ligation, cDNA Library Assay, Sequencing

Journal: Nature methods

Article Title: Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

doi: 10.1038/s41592-023-02028-1

Figure Lengend Snippet: a, Metagene average profiles around the start (left) and stop (right) codons from sucrose cushion purified yeast RPFs generated by P1 nuclease (top, red) or RNase I (bottom, blue) digestion. The 5′ ends of aligned reads were counted, and counts for each gene were rescaled by the mean codon count for the gene, excluding those RPFs that are aligned to the first 15 and last 10 codons, prior to averaging. b, As in (a), for sucrose cushion purified human 293T cell RPFs generated by P1 nuclease (top, red) or RNase I (bottom, blue) digestion. c, Gene-level ribosome occupancy estimates from sucrose cushion purified yeast RPFs generated by P1 nuclease and RNase I digestion. Read counts for each gene were normalized by DESeq2. d, Gene-level estimates from sucrose cushion purified human 293T cell RPFs generated by P1 nuclease and RNase I digestion. Read counts for each gene were normalized by DESeq2. e, Average profile of yeast footprints at start codons for P1 nuclease (red, 30 – 40 nt) and RNase I (blue, 25 – 29 nt) libraries. Footprint alignments were counted separately for each gene monitoring read length as well as 5′ end position (left) and 3′ end position (right), then averaged as in (a). A heatmap shows footprint abundance according to length and end position (below), and the end position average summed across all lengths is shown (above each heatmap matrix. The 5′ and 3′ end averages are shown to the left and to the right, respectively, of a black vertical bar. A diagram of a translating ribosome footprint (top) indicates mRNA cleavage positions of P1 nuclease (red triangle) and RNase I (blue triangle). f, Average profile of human cell footprints at start codons for P1 nuclease (red, 33 – 40 nt) and RNase I (blue, 27 – 32 nt) libraries, as in (e). g-h, Comparison codon-level ribosome occupancy estimates from (g) yeast or (h) human cell RPFs generated by P1 nuclease and RNase I digestion, as in Fig. 1c. In (h), arginine codons are shown in red. i, Schematic of proposed P1 nuclease and RNase I cleavage sites around an mRNA-engaged ribosome. Increased frequency of an RPF terminal position is indicated by increasing color saturation.

Article Snippet: Anecdotally, we advise RNase I RPFs be first subjected to Oligo Clean and Concentrate (Zymo Research) purification before T4-PNK dephosphorylation.

Techniques: Purification, Generated, Comparison

Journal: Genome Biology

Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

doi: 10.1186/gb-2012-13-3-r23

Figure Lengend Snippet: Performance evaluation of five rRNA depletion methods. (a) Shown is the distribution of RNA-seq reads aligning to protein-coding sequences (CDS; blue), rRNA (red), and other regions (tRNA, non-coding RNA, small RNA, and intergenic regions; gray) for undepleted total RNA (top) and five rRNA depletion protocols. (b) The lengths of the black bars represent the coefficient of determination (R2) for RPKM values before and after rRNA depletion using different rRNA-depletion methods. Ribo-Zero, normalization using duplex-specific nuclease (DSN) and Ovation were tested on a 1:1:1 pool (by mass) of total RNA prepared from P. marinus, E. coli, and R. sphaeroides. MICROBExpress and mRNA-ONLY were performed on individual RNA preparations without pooling.

Article Snippet: The second strand was synthesized by adding 1× of second strand buffer (5×; Invitrogen), 0.2 mM of dNTPs (10 mM; Invitrogen), 40 U of E. coli DNA polymerase I (10 U/μl; NEB, Ipswich, MA, USA), 10 U of E. coli DNA ligase (10 U/μl; NEB), 5 U of RNase H (5 U/μl; Invitrogen) to the first strand reaction (150 μl total volume).

Techniques: RNA Sequencing Assay

Journal: Genome Biology

Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

doi: 10.1186/gb-2012-13-3-r23

Figure Lengend Snippet: Depletion of rRNA in a mixture of total RNAs from E. coli, R. sphaeroides and P. marinus with Ribo-Zero is reproducible and works well with fragmented total RNA. (a) The pie charts represent the mapped read distributions of protein-coding genes (CDS; blue), rRNA (red), and other reads (tRNA, non-coding RNA, small RNA and intergenic regions; gray) for undepleted total RNA, two technical replicates of Ribo-Zero treatment of intact total RNA and for Ribo-Zero treatment of fragmented total RNA. (b,c) Double-log scatter plots of RPKM values and the coefficient of determination (R2) for the technical Ribo-Zero replicates (b) and for Ribo-Zero treatment of fragmented versus intact total RNA (c). Points on the axes represent CDSs with zero coverage in one of the two samples. The number of data points in the diagonal cloud and on the axes is indicated. The total number of annotated CDSs in the three bacterial genomes is 10,278.

Article Snippet: The second strand was synthesized by adding 1× of second strand buffer (5×; Invitrogen), 0.2 mM of dNTPs (10 mM; Invitrogen), 40 U of E. coli DNA polymerase I (10 U/μl; NEB, Ipswich, MA, USA), 10 U of E. coli DNA ligase (10 U/μl; NEB), 5 U of RNase H (5 U/μl; Invitrogen) to the first strand reaction (150 μl total volume).

Techniques:

Journal: Genome Biology

Article Title: Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes

doi: 10.1186/gb-2012-13-3-r23

Figure Lengend Snippet: Strand specificity of RNA-seq reads. Shown is a 17-kb window of the E. coli genome viewed with the Artemis browser [28]. The mapped reads aligning to the top strand (green) or bottom stand (purple) consistent with the direction of the annotated genes as represented by the blue boxes with arrows and corresponding gene ID numbers and operons below (for example, genes b3196 through b3206).

Article Snippet: The second strand was synthesized by adding 1× of second strand buffer (5×; Invitrogen), 0.2 mM of dNTPs (10 mM; Invitrogen), 40 U of E. coli DNA polymerase I (10 U/μl; NEB, Ipswich, MA, USA), 10 U of E. coli DNA ligase (10 U/μl; NEB), 5 U of RNase H (5 U/μl; Invitrogen) to the first strand reaction (150 μl total volume).

Techniques: RNA Sequencing Assay

Journal: bioRxiv

Article Title: PRMT7 ablation stimulates anti-tumor immunity and sensitizes melanoma to immune checkpoint blockade

doi: 10.1101/2021.07.28.454202

Figure Lengend Snippet: (A) RT-qPCR analysis of selected retrotransposons, IFNs and ISGs transcripts in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells. Bar graphs represent the mean fold-change ± SD. Data are representative of three independent experiments. Statistical significance was calculated by unpaired student t test (* p <0.1; ** p <0.01; *** p <0.001; **** p <0.0001). (B) The assessment of both sense and antisense transcripts of selected ERVs ( MuERV-L and IAP ) using strand-specific primers for RT-PCR (TASA-TD technique) in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells. β-actin was used as a negative control for antisense transcription. A representative experiment is shown of three independent experiments. (C) dsRNA enrichment of MuERV-L IAP, MuSD and Line-1 retrotransposons in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells by RT-qPCR analysis following RNase A treatment. (D) Total RNA extracted from sgCTL and sgPRMT7-1 B16 cells were treated with Mock, RNase III or RNase A (under high salt condition: 350 mM NaCl), dotted on Hybond N+ membrane and immunoblotted with the J2 antibody and visualized by methylene for loading control. Dots are denoted by numbers: 1, 3, 5 for sgCTL and 2, 4, 6 for sgPRMT7-1 cells nontreated (dots 1 and 2), treated with RNase III (dots 3 and 4) or RNase A (dots 5 and 6). (E) sgCTL and sgPRMT7-1 B16 cells were incubated with 0.5 mM sodium arsenite for 1h or 45°C (heat shock) treatment for 30 min. Cells were then fixed with 4% PFA and immunostained using anti-G3BP1 antibodies. A representative IF image is shown 60x magnification. DAPI, 4’,6-diamidino-2-phenylindole, was shown in blue as indicated. (F) The average number of SGs per cell of the staining done in (E) was quantified using image J software and presented as a bar plot ( n =60 to 70 cells per condition). Bar graphs show mean intensity ± SEM. Statistical significance was calculated by unpaired student t test (**** p <0.0001). (G) RT-qPCR analysis of DNMT mRNAs ( Dnmt1, Dnmt3a and Dnmt3b ) in sgCTL, sgPRMT7 B16 melanoma cells. Bar graphs represent the mean fold-change ± SD. Data are representative of three independent experiments. Statistical significance was calculated by unpaired student t test (**** p <0.0001). (H) Immunoblot of DNMT proteins (DNMT1, DNMT3a and DNMT3b) in sgCTL, sgPRMT7-1 and sgPRMT7-2 B16 cells. β-actin was used as the loading control. A representative experiment is shown out of three independent experiments. The molecular mass markers are indicated in the left in kDa. The DNMT bands are shown with arrowheads.

Article Snippet: 5 µg of total RNA extracted from B16.F10 cells was dissolved in 46 µl H2O and digested with 1U RNase A (Ambion #AM2270) under high salt condition: 3.5 µl NaCl (5 M stock) to a total volume of 50 µl and mixed well, followed by incubation for 30 min at 37°C.

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Negative Control, Incubation, Staining, Software, Western Blot