Journal: Frontiers in Genetics

Article Title: Depletion of Hemoglobin Transcripts and Long-Read Sequencing Improves the Transcriptome Annotation of the Polar Bear ( Ursus maritimus )

doi: 10.3389/fgene.2019.00643

Figure Lengend Snippet: Schematic of Long-DASH, (A) Whole blood RNA is extracted, and full-length cDNA is generated with the first half of the Smart-seq2 protocol. The cDNA is then depleted of hemoglobin transcripts using the recombinant S. pyogenes Cas9 protein bound to hemoglobin-specific sgRNA, which cuts hemoglobin cDNA molecules by introducing double-strand breaks (△) in a sequence-specific manner. The cut molecules can no longer be exponentially amplified with PCR, so a subsequent PCR step is performed to enrich for complete non-hemoglobin cDNA molecules. The resulting hemoglobin-depleted cDNA pool is then sequenced using the ONT-based R2C2 library prep and the Illumina-based Smart-seq2 library prep.

Article Snippet: As a proof of concept, we evaluated three hemoglobin-depleted and non-depleted polar bear whole blood transcriptomes using our ONT-based R2C2 ( ) full-length cDNA sequencing method and an Illumina-based modified Smart-seq2 method.

Techniques: Generated, Recombinant, Sequencing, Amplification

Journal: Frontiers in Genetics

Article Title: Depletion of Hemoglobin Transcripts and Long-Read Sequencing Improves the Transcriptome Annotation of the Polar Bear ( Ursus maritimus )

doi: 10.3389/fgene.2019.00643

Figure Lengend Snippet: Long-DASH depletes hemoglobin from cDNA. (A) Hemoglobin content was measured in Smart-seq2 (Illumina) libraries of depleted (blue) or undepleted (black) cDNA pools. (B) (Top) and (C) Scatterplots comparing gene expression in undepleted and depleted Smart-seq2 libraries of PB3, PB19, and PB21 with reads aligning to hemoglobin loci (red) either included (B) or excluded (C) . (B) (Bottom) Scatterplots showing log2(fold-change) between depleted and undepleted cDNA pools as calculated by (depleted [log2(RPM+1)]-undepleted [log2(RPM+1)]) with hemoglobin loci included in the RPM normalization.

Article Snippet: As a proof of concept, we evaluated three hemoglobin-depleted and non-depleted polar bear whole blood transcriptomes using our ONT-based R2C2 ( ) full-length cDNA sequencing method and an Illumina-based modified Smart-seq2 method.

Techniques: Expressing

Journal: Frontiers in Genetics

Article Title: Depletion of Hemoglobin Transcripts and Long-Read Sequencing Improves the Transcriptome Annotation of the Polar Bear ( Ursus maritimus )

doi: 10.3389/fgene.2019.00643

Figure Lengend Snippet: R2C2/Mandalorion isoforms identify new features in the polar bear transcriptome. (Top) General workflow for comparing RefSeq mRNAs and Mandalorion isoforms is shown on the left. RefSeq mRNAs were aligned to the polar genomes using minimap2 and converted to GTF format to create a reference annotation. Isoforms determined by Mandalorion were then classified using this reference annotation using the sqanti_qc algorithm. Isoforms were classified as Novel_not_in_catalog (NNC), Novel_in_catalog (NIC), Full_splice_match (FSM), Incomplete_splice_match (ISM), and Intergenic (IG). New transcriptome features were then determined based on the minimap2 alignments of isoforms in the indicated categories. (Bottom) R2C2 and Smart-seq2 read coverage around newly identified TSS, the splice sites (3’ and 5’) of newly identified exons, and newly identified polyA sites.

Article Snippet: As a proof of concept, we evaluated three hemoglobin-depleted and non-depleted polar bear whole blood transcriptomes using our ONT-based R2C2 ( ) full-length cDNA sequencing method and an Illumina-based modified Smart-seq2 method.

Techniques:

Journal: Biology of Reproduction

Article Title: What has single-cell RNA-seq taught us about mammalian spermatogenesis?

doi: 10.1093/biolre/ioz088

Figure Lengend Snippet: Mouse spermatogenesis single-cell RNA-seq datasets.

Article Snippet: scRNA-seq Chemistry/Method , 3′ sequencing and full length sequence (SMART-seq2) , 3′ sequencing and barcoding (10x Genomics) , 3′ sequencing and barcoding (Oliginal Drop-seq) , 3′ sequencing and barcoding (10x Genomics) , 3′ sequencing and barcoding (SMART-seq2 and Microwell-seq) , full length sequence (Fluidigm C1), 3′ sequencing and barcoding (10x Genomics) , 3′ sequencing and barcoding (10x Genomics) , 3′ sequencing and barcoding (10x Genomics) , full length sequence (Fluidigm C1) , full length sequence (Fluidigm C1) , 3′ sequencing and barcoding (10x Genomics) , 3′ sequencing and barcoding (10x Genomics) , full length sequence (Fluidigm C1).

Techniques: Knock-Out, Transplantation Assay, Sequencing

Journal: Biology of Reproduction

Article Title: What has single-cell RNA-seq taught us about mammalian spermatogenesis?

doi: 10.1093/biolre/ioz088

Figure Lengend Snippet: Human spermatogenesis single-cell RNA-seq datasets.

Article Snippet: scRNA-seq Chemistry/Method , 3′ sequencing and full length sequence (SMART-seq2) , 3′ sequencing and barcoding (10x Genomics) , 3′ sequencing and barcoding (Oliginal Drop-seq) , 3′ sequencing and barcoding (10x Genomics) , 3′ sequencing and barcoding (SMART-seq2 and Microwell-seq) , full length sequence (Fluidigm C1), 3′ sequencing and barcoding (10x Genomics) , 3′ sequencing and barcoding (10x Genomics) , 3′ sequencing and barcoding (10x Genomics) , full length sequence (Fluidigm C1) , full length sequence (Fluidigm C1) , 3′ sequencing and barcoding (10x Genomics) , 3′ sequencing and barcoding (10x Genomics) , full length sequence (Fluidigm C1).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Depletion of hemoglobin transcripts and long read sequencing improves the transcriptome annotation of the polar bear ( Ursus maritimus )

doi: 10.1101/527978

Figure Lengend Snippet: A) Whole Blood RNA is extracted and full-length cDNA is generated with the first half of the Smart-seq2 protocol. The cDNA is then depleted of hemoglobin transcripts using the recombinant S. pyogenes Cas9 protein bound to hemoglobin specific sgRNA which cuts hemoglobin cDNA molecules by introducing double strand breaks (△) in a sequence specific manner. The cut molecules can no longer be exponentially amplified with PCR, so a subsequent PCR step is performed to enrich for complete non-hemoglobin cDNA molecules. The resulting hemoglobin depleted cDNA pool is then sequenced using the ONT-based R2C2 library prep and the Illumina-based Smart-seq2 library prep.

Article Snippet: Using this method, we sequenced depleted full-length cDNA in parallel using both our Oxford Nanopore Technology (ONT) based R2C2 long-read approach, as well as the Illumina short-read based Smart-seq2 approach.

Techniques: Generated, Recombinant, Sequencing, Amplification

Journal: bioRxiv

Article Title: Depletion of hemoglobin transcripts and long read sequencing improves the transcriptome annotation of the polar bear ( Ursus maritimus )

doi: 10.1101/527978

Figure Lengend Snippet: Length distribution of R2C2 consensus reads is shown as swarmplots in the indicated samples. Independent Long-DASH replicates (R1 and R2) were performed for samples PB3 and PB19 but not PB21. Percent of Hemoglobin reads as determined with a kmer approach is given in red on top.

Article Snippet: Using this method, we sequenced depleted full-length cDNA in parallel using both our Oxford Nanopore Technology (ONT) based R2C2 long-read approach, as well as the Illumina short-read based Smart-seq2 approach.

Techniques:

Journal: bioRxiv

Article Title: Depletion of hemoglobin transcripts and long read sequencing improves the transcriptome annotation of the polar bear ( Ursus maritimus )

doi: 10.1101/527978

Figure Lengend Snippet: Genome Browser views of the RBX1 locus (A), the GMFG locus (B), a locus likely corresponding to the CCDC72 gene not yet included in the RefSeq mRNA set (C). From top to bottom, 1) RefSeq mRNAs alignments, 2) new features based on Mandalorion isoforms (green: TSS, red: polyA site, blue: new exon or locus), 3) Mandalotion isoforms, and 4) R2C2 reads. Plus strand alignment are in blue, minus strand alignments in orange.

Article Snippet: Using this method, we sequenced depleted full-length cDNA in parallel using both our Oxford Nanopore Technology (ONT) based R2C2 long-read approach, as well as the Illumina short-read based Smart-seq2 approach.

Techniques: