smart-seq2 library preparation  (Illumina Inc)

Journal: eLife

Article Title: The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation

doi: 10.7554/eLife.72377

Figure Lengend Snippet: ( A ) Experimental design. The experiment was performed once, using three biological replicates (independent lines) per group (genotype). ( B ) Expression pattern of ECT2-mCherry in root tips of ect2-1 ECT2-mCherry and te234 ECT2 W464A -mCherry genotypes by fluorescence microscopy. ( C ) Protein blot showing expression levels of ECT2-mCherry in the 3+3 lines of ect2-1 ECT2-mCherry and te234 ECT2 W464A -mCherry used as biological replicates for FACS selection of ECT2-expressing cells. Amido black (A.B.) is used as loading control. ( D ) Fluorescence profile (mCherry vs. GFP fluorescence) of root cells (protoplasts) from the transgenic lines in ( C ). The complete set of lines/samples is shown in . Non-transgenic Col-0 WT is shown as control for background autofluorescence. Cells with a fluorescence profile within the outlined areas were selected for RNA extraction, Smart-seq2 library construction, and sequencing. ( E ) Genes with more than one poly(A) site cluster (PAC) in the different target/non-target sets. Dark shades are genes in which the dominant PAC in te234 ECT2 W464A -mCherry samples differs from the one in ect2-1 ECT2-mCherry . ( F, G ) Distribution of distances (d [nt]) of the most common poly(A) site between te234 ECT2 W464A -mCherry and ect2-1 ECT2-mCherry samples for all genes where the most common poly(A) site could be determined in both genotypes (6648 non-targets, 4072 permissive targets, and 1486 stringent targets). Negative values are upstream (5′) and positive values are downstream (3′) relative to the gene orientation. ( F ) Distances are binned by ±10, ±100, ±1000, and >1000 bp. ( G ) Distances are plotted by nucleotide in a ±40 bp window. Figure 5—source data 1. Original (uncropped) membrane from .

Article Snippet: Commercial assay or kit , Illumina DNA Nextera Flex kit (now called Illumina DNA Prep) , Illumina , Cat. # 20018704 , Used for Smart-seq2 library preparation.

Techniques: Expressing, Fluorescence, Microscopy, Selection, Transgenic Assay, RNA Extraction, Sequencing

Journal: eLife

Article Title: The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation

doi: 10.7554/eLife.72377

Figure Lengend Snippet: Fluorescence profile (mCherry vs. GFP fluorescence) of root cells (protoplasts) from the transgenic lines used as replicates for transcriptomic analyses ( and ). Non-transgenic Col-0 WT is shown as control for background autofluorescence. Cells with a fluorescence profile within the outlined areas were selected for RNA extraction, Smart-seq2 library construction, and sequencing.

Article Snippet: Commercial assay or kit , Illumina DNA Nextera Flex kit (now called Illumina DNA Prep) , Illumina , Cat. # 20018704 , Used for Smart-seq2 library preparation.

Techniques: Fluorescence, Transgenic Assay, RNA Extraction, Sequencing

Journal: eLife

Article Title: The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation

doi: 10.7554/eLife.72377

Figure Lengend Snippet: ( A ) Summary of poly(A)-containing reads (i.e., reads with at least nine untemplated As) after removal of reads mapping upstream of purine-rich sites. These reads were used for cluster identification, yielding 17,028 putative poly(A) site clusters (PACs), from which 14,667 were retained after further filtering of potential false positives (see Materials and methods). ( B ) Features of PACs. (Upper panel) Genomic distance between most upstream and most downstream poly(A) sites within each cluster (median length of 105 bp). (Lower panel) Total number of genomic positions within the cluster where at least one read with an untemplated poly(A) tail was detected (median of 12 poly(A) sites per cluster). ( C ) PACs sorted by ECT2/3 target status. Percentages of genes with more than one PAC refer to the number of genes with PACs. Percentages of genes with a dominant PAC (defined as the cluster with the most reads) that is different between te234/ECT2 W464A -mCherry and ect2-1/ECT2-mCh samples refer to the number of genes with more than one PAC. ( D ) Variation in dominant polyadenylation sites for the non-ECT2/3-targeted transcript LAX3 , an example of gene with different dominant PAC. Independently of the fact that the total amount of poly(A) reads is generally higher in te234/ECT2 W464A -mCherry compared to ect2-1/ECT2-mCh samples (notice that the scales have been adjusted for optimal comparison of PAC usage within samples), the ratio between the number of reads in the upstream and the downstream clusters is different in the two genotypes. Transcript annotation is based on TAIR10. ( E ) Mean TPMs (Smart-seq2 data of sorted protoplasts, combining all six samples) of genes in the different ECT2/3 targets groups (upper panel). The significantly lower likelihood for ECT2/3 targets to have a different dominant PAC upon loss of ECT2/3/4 function depletion compared to non-targets could be due to differences in transcript abundance between the target and non-target groups. Looking at only the 2200 most highly expressed non-target genes, only 5.5% of these genes have a different dominant PAC in te234/ECT2 W464A -mCherry than ect2-1/ECT2-mCh samples (lower panel, dark shading refers to genes with different dominant PAC as in ), significantly smaller than the percentage for all non-target genes (20.8%, ) (p=3.2e−9, Fisher’s exact test).

Article Snippet: Commercial assay or kit , Illumina DNA Nextera Flex kit (now called Illumina DNA Prep) , Illumina , Cat. # 20018704 , Used for Smart-seq2 library preparation.

Techniques:

Journal: eLife

Article Title: The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation

doi: 10.7554/eLife.72377

Figure Lengend Snippet: ( A ) Principal component analysis of transcriptome expression values (TPM) in Smart-seq2 libraries from FACS-sorted root protoplasts of te234/ECT2 W464A -mCherry and ect2-1/ECT2-mCherry 5-day-old seedlings (left panel), or in RNA-seq libraries obtained from root tips of te234 and wild type 4-day-old seedlings (right panel). ( B ) Boxplots of log 2 fold change expression values between the genotype-pairs described in ( A ). ( C ) Volcano plots showing genes differentially expressed between the genotype-pairs described in ( A ). Upregulated non-ECT2/3 targeted transcription factors and stress-responsive genes are marked. ( D, E ) List with the 10 most significantly enriched GO terms among significantly upregulated ECT2/3 targets (permissive set) ( D ), or downregulated non-targets ( E ) in root tips of te234 compared to wild type. GO, gene ontology.

Article Snippet: Commercial assay or kit , Illumina DNA Nextera Flex kit (now called Illumina DNA Prep) , Illumina , Cat. # 20018704 , Used for Smart-seq2 library preparation.

Techniques: Expressing, RNA Sequencing Assay

Journal: eLife

Article Title: The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation

doi: 10.7554/eLife.72377

Figure Lengend Snippet: ( A ) Scatterplot of TPM expression values in Smart-seq2 libraries of root protoplasts expressing ECT2-mCherry in te234/ECT2 W464A -mCherry versus ect2-1/ECT2-mCherry samples. ( B ) Volcano plots reveal genes differentially expressed between the genotypes described in ( A ). ( C ) Boxplots of log 2 fold change expression values between te234/ECT2 W464A -mCherry and ect2-1/ECT2-mCherry samples. ( D, E ) Bar plots showing the amount of significantly upregulated and downregulated genes in ECT2/3 targets and non-targets. ( F, G ) List with the 10 most significantly enriched GO terms among significantly downregulated ECT2/3 targets (permissive set) ( F ), or upregulated non-targets ( G ) upon loss of ECT2/3/4 function. GO, gene ontology.

Article Snippet: Commercial assay or kit , Illumina DNA Nextera Flex kit (now called Illumina DNA Prep) , Illumina , Cat. # 20018704 , Used for Smart-seq2 library preparation.

Techniques: Expressing

Journal: eLife

Article Title: The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation

doi: 10.7554/eLife.72377

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Illumina DNA Nextera Flex kit (now called Illumina DNA Prep) , Illumina , Cat. # 20018704 , Used for Smart-seq2 library preparation.

Techniques: Selection, Transgenic Assay, Sequencing, Clone Assay, Software, Fluorescence