Journal: Scientific reports

Article Title: A cost-effective RNA sequencing protocol for large-scale gene expression studies.

doi: 10.1038/srep09570

Figure Lengend Snippet: Figure 2 | Performance and cost of LM-seq protocol. (A). Scatter plot of TPM values of two technical replicates of LM-Seq of hES cells using 100 ng total RNA as starting material. Pearson correlation is reported on top of the plot. (B). Scatter plot of TPM values of LM-Seq and TruSeq of hES cells using 100 ng total RNA as starting material. Spearman’s rank correlation (r) and Pearson correlation are reported on top of the plot. (C). Average reads distribution across all transcripts. Blue: LM-Seq, Red: TruSeq. (D). The comparison of the percentage of reads that map to the correct strands of the human transcriptome between data generated by LM-Seq and TruSeq Stranded mRNA-Seq protocol (error bar: 1/2standard deviation). (E). Comparison of preparation cost per sample between commercially available kits (NEBNext Ultra Directional RNA Library Prep Kit from NEB, ScriptSeq Complete Kit from Epicentre, SMARTer Stranded RNA-seq Kit from Clontech, TruSeq Stranded mRNA Kit from Illumina) and LM-Seq.

Article Snippet: Sultan, M. et al. A simple strand-specific RNA-Seq library preparation protocol combining the Illumina TruSeq RNA and the dUTP methods.

Techniques: Comparison, Generated, RNA Sequencing

Journal: Genome Biology

Article Title: Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated dCas9

doi: 10.1186/s13059-020-01973-w

Figure Lengend Snippet: Multiplexed CAPTURE of locus-specific long-range DNA interactions. a Schematic of multiplexed analysis of locus-specific chromatin interactions by the redesigned CAPTURE2.0 system containing the C-terminal biotin-tagged dCas9 (dCas9-CBio) and sgRNA. Major steps of the CAPTURE-3C-seq method are shown. b Schematic of dCas9-mediated multiplexed capture of the human β-globin LCR. c Genome-wide analysis of dCas9 binding in cells expressing LCR-targeting sgRNAs (sgLCR) or non-targeting sgGal4. Data points for the sgRNA target regions are shown by arrowheads, and the predicted off-targets are shown as red dots. The x - and y -axes denote the log2 mean read counts and the log2 ratio of read counts in sgLCR and sgGal4 samples from N = 2 and 4 CAPTURE-ChIP-seq experiments, respectively. d Genome-wide differential gene expression analysis was performed using RNA-seq in K562 cells expressing dCas9-CBio with sgLCR or wild-type (WT) K562 cells. The β-like globin genes are indicated by colored data points. Pearson correlation coefficient ( R ) value is shown ( N = 2 RNA-seq experiments). e Browser view of LCR-mediated long-range interactions (chr11: 5,222,424-5,323,623; hg19) is shown. Contact profiles including the density map and interactions (or loops) for the dCas9-captured LCR or the resolved individual HS regions are shown. The statistical significance of interactions was determined by the Bayes factor (BF) and indicated by the color scale bars. DHS, ChIP-seq, RNA-seq, ChromHMM, CAPTURE-ChIP-seq (sgLCR), and ChIA-PET (CTCF and RNAPII) data are shown for comparison

Article Snippet: Human K562 cells were obtained from ATCC and cultured in an IMDM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: 3C-Seq, Genome Wide, Binding Assay, Expressing, ChIP-sequencing, Gene Expression, RNA Sequencing, ChIA Pet Assay, Comparison

Journal: Genome Biology

Article Title: Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated dCas9

doi: 10.1186/s13059-020-01973-w

Figure Lengend Snippet: Multiplexed CAPTURE of erythroid super-enhancers. a Schematic of the multiplexed analysis of erythroid SEs. SEs were identified by ROSE using H3K27ac ChIP-seq signal in K562 cells. Schematic of sgRNA design is shown. Pie charts on the left show the distribution of the captured SEs and constituent enhancers at intragenic, intergenic, or both regions. Pie charts on the right show the numbers and percentages of captured SEs and constituent enhancers (green color). b Genome-wide analysis of dCas9 binding in cells expressing SE-targeting sgRNAs (sgSE) or non-targeting sgGal4. Data points for the sgRNA captured SEs are shown as green. The x - and y -axes denote the log2 mean read counts and the log2 ratio of read counts in sgSE and sgGal4 samples from N = 2 and 4 CAPTURE-ChIP-seq experiments, respectively. c Genome-wide differential gene expression analysis was performed using RNA-seq in K562 cells expressing dCas9-CBio with sgSE or WT K562 cells. Data points for SE target genes and other genes are shown as red and gray, respectively. Pearson correlation coefficient ( R ) value is shown ( N = 2 RNA-seq experiments). d Analysis of SE-mediated long-range interactions by categorizing all interactions into SEs to gene promoters (SE-P), SEs to gene bodies (SE-G), and SEs to other genomic regions (SE-O). Schematic of SE-mediated interactions is shown on the top. The interaction frequency was calculated by the normalized PETs per kilobase of captured DNA sequences. Boxes show the median of the data and quartiles, and whiskers extend to 1.5× of the interquartile range. P values were calculated by a two-sided Kolmogorov-Smirnov (K-S) test. e Pie chart shows the fractions of the captured SE-mediated interactions between SEs and gene targets. f A representative locus is shown for the single SE to single gene interactions (SE137). Contact profiles including the density map and interactions for the dCas9-captured SE region (red bar) are shown. The statistical significance of interactions was determined by the Bayes factor (BF) and indicated by the color scale bars. DHS, ChIP-seq, ChromHMM, ChIA-PET (CTCF and RNAPII), and in situ Hi-C data are shown for comparison. g A representative locus is shown for the single SE to multiple genes (SE43). h A representative locus is shown for the multiple SEs to multiple genes (SE41 and SE42)

Article Snippet: Human K562 cells were obtained from ATCC and cultured in an IMDM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: ChIP-sequencing, Genome Wide, Binding Assay, Expressing, Gene Expression, RNA Sequencing, ChIA Pet Assay, In Situ, Hi-C, Comparison

Journal: Genome Biology

Article Title: Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated dCas9

doi: 10.1186/s13059-020-01973-w

Figure Lengend Snippet: Hierarchical organization of super-enhancers identified by multiplexed CAPTURE. a Schematic of the hierarchical structure of SEs based on constituent enhancer-mediated long-range chromatin interactions. b Identification of hierarchical SEs by the H-score computational metric. c A representative locus is shown for a hierarchical SE containing the hub enhancer. Contact profiles including the density map, interactions between enhancers, and all interactions for the dCas9-captured SE region (red bar) are shown. The identified hub and non-hub enhancers are depicted by green (hub) and red (non-hub) lines, respectively. d A representative locus is shown for a non-hierarchical SE without hub enhancer. e Chromatin landscapes at hub, non-hub enhancers, and non-hierarchical SEs in K562 cells. Spatial distribution of DHS, histone marks (H3K4me1 and H3K27ac), TFs (p300, GATA1, and TAL1), RNAPII, and chromatin structure factors (CTCF, SMC3, and RAD21) is shown in hub ( N = 42) and non-hub enhancers ( N = 260) and non-hierarchical SEs ( N = 117). P values were calculated using the two-sample Kolmogorov-Smirnov (K-S) test. *** P < 0.001, ** P < 0.01, * P < 0.05, n.s. not significant

Article Snippet: Human K562 cells were obtained from ATCC and cultured in an IMDM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: