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: Cell Rep Total cell number 1204 53 510 (from 2 P5, 1 P10, 1 P15, 1 P20, 1 P25, 1 P30, 1 P35, 2 8–9 week mice) 34 633 1200–2500 per sample (from 1 P6, 1 P14, 1 P18, 1 P25, 1 P30, 1 8week mice) 19 659 (>50 mice) 31 163 (from 21 adult mice, 30 P6 mice) 62 600 (from 11 to 38 week WT, Mlh3 KO, Cul4a KO, Hormad1 KO, and Cnp mice) Not mentioned 71 175 (80 from P1.5, 48 from P3.5, and 47 from P5.5) Interstitial steroidogenic progenitor cells (6362 cells), SCs (1932 cells), fetal Leydig cells (521 cells), primordial germ cells (483 cells), and endothelial cells (180 cells) from E16.5 2500 (1250 each from 2 mice) 181 (71 from P3 WT, 53 from P7 WT, 57 from Rhox10 KO P3) First-Wave + + – + – + – – + + – – + Unselected steady-state spermtogenesis – + + + – + + – – – – + – Sorted Spermatogonia + – + + – + + + – – – – – Sorted Spermatocytes + – + + – + + – – – – – – Sorted Spermatids + – + + – + + – – – – – – Somatic cells – Sertoli cells, Leydig cells, Myoid cells, Endothelial cells, Marcrophages Sertoli cells, Leydig cells, Myoid cells, Endothelial cells, Macrophage, Inntate Lymphosid Sertoli cells, Endothelial, Hematopoietic cells, Smooth muscle – Sertoli cells, Peritubular cells (adult) Sertoli cells, Leydig cells – – Sertoli cells Endothelial cells, fetal Leydig cells, Sertoli cells Sertoli cells, Leydig cells – Validation methods Knockout, ChIP-seq RNA scope, ChIP-seq IHC, smFISH IHC – IHC, qRT-PCR Knockout, IHC Knockout, Transplantation, Bulk RNA-seq, IHC IHC, Function (RA inhibitior) Knockout, ChIP-qPCR, IHC, WB Knockout in Sertoli cells – Knockout, IHC 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) Replication Replication unclear 1 replicate (P10, P15, P20, P25, P30, and P35) and 2 biological replicates (P5 and adult) 6 x samples from individual adult mice.

Techniques: Biomarker Discovery, Knock-Out, RNAscope, 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: Cell Rep Total cell number 1204 53 510 (from 2 P5, 1 P10, 1 P15, 1 P20, 1 P25, 1 P30, 1 P35, 2 8–9 week mice) 34 633 1200–2500 per sample (from 1 P6, 1 P14, 1 P18, 1 P25, 1 P30, 1 8week mice) 19 659 (>50 mice) 31 163 (from 21 adult mice, 30 P6 mice) 62 600 (from 11 to 38 week WT, Mlh3 KO, Cul4a KO, Hormad1 KO, and Cnp mice) Not mentioned 71 175 (80 from P1.5, 48 from P3.5, and 47 from P5.5) Interstitial steroidogenic progenitor cells (6362 cells), SCs (1932 cells), fetal Leydig cells (521 cells), primordial germ cells (483 cells), and endothelial cells (180 cells) from E16.5 2500 (1250 each from 2 mice) 181 (71 from P3 WT, 53 from P7 WT, 57 from Rhox10 KO P3) First-Wave + + – + – + – – + + – – + Unselected steady-state spermtogenesis – + + + – + + – – – – + – Sorted Spermatogonia + – + + – + + + – – – – – Sorted Spermatocytes + – + + – + + – – – – – – Sorted Spermatids + – + + – + + – – – – – – Somatic cells – Sertoli cells, Leydig cells, Myoid cells, Endothelial cells, Marcrophages Sertoli cells, Leydig cells, Myoid cells, Endothelial cells, Macrophage, Inntate Lymphosid Sertoli cells, Endothelial, Hematopoietic cells, Smooth muscle – Sertoli cells, Peritubular cells (adult) Sertoli cells, Leydig cells – – Sertoli cells Endothelial cells, fetal Leydig cells, Sertoli cells Sertoli cells, Leydig cells – Validation methods Knockout, ChIP-seq RNA scope, ChIP-seq IHC, smFISH IHC – IHC, qRT-PCR Knockout, IHC Knockout, Transplantation, Bulk RNA-seq, IHC IHC, Function (RA inhibitior) Knockout, ChIP-qPCR, IHC, WB Knockout in Sertoli cells – Knockout, IHC 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) Replication Replication unclear 1 replicate (P10, P15, P20, P25, P30, and P35) and 2 biological replicates (P5 and adult) 6 x samples from individual adult mice.

Techniques: Biomarker Discovery, Sequencing

Journal: NAR Cancer

Article Title: Single-cell multi-gene identification of somatic mutations and gene rearrangements in cancer

doi: 10.1093/narcan/zcad034

Figure Lengend Snippet: Cancer samples used for single-cell adaptive sequencing

Article Snippet: The libraries were prepared with 900 fmol of pooled amplicon for PromethION Flow Cell FLO-PRO002 (Oxford Nanopore Technologies) using Native Barcoding Expansion and Ligation Sequencing Kit (Oxford Nanopore Technologies) as per the manufacturer’s protocol.

Techniques: Mutagenesis, Variant Assay, CRISPR, Sequencing, Transduction, Amplification

Journal: NAR Cancer

Article Title: Single-cell multi-gene identification of somatic mutations and gene rearrangements in cancer

doi: 10.1093/narcan/zcad034

Figure Lengend Snippet: An adaptive sampling method for sequencing target cDNAs from scRNA-seq. ( A ) Overview of single-cell library preparation, long- and short-read sequencing analysis, and integration of results from both modalities. Integrative Genomics Viewer (IGV) screenshot of SRSF5 targeting sites from cells with gRNAs: ( B ) SRSF5-1 and ( C ) SRSF5-2. ( D ) Boxplot showing CRISPR-induced mutation rate for all genes targeted.

Article Snippet: The libraries were prepared with 900 fmol of pooled amplicon for PromethION Flow Cell FLO-PRO002 (Oxford Nanopore Technologies) using Native Barcoding Expansion and Ligation Sequencing Kit (Oxford Nanopore Technologies) as per the manufacturer’s protocol.

Techniques: Sampling, Sequencing, CRISPR, Mutagenesis

Journal: NAR Cancer

Article Title: Single-cell multi-gene identification of somatic mutations and gene rearrangements in cancer

doi: 10.1093/narcan/zcad034

Figure Lengend Snippet: Single-cell mutations from the T1 and T2 appendiceal cancers. ( A ) Location of tumor samples for patient 8605, and variants detected from clinical diagnostic sequencing having sufficient long-read depth for analysis. ( B ) IGV screenshots for T1 alignments, covering the lengths of KRAS and GNAS genes. ( C ) UMAP clustered plot showing integration of T1 and T2 samples. ( D ) UMAP clustered plot annotated with cell types and dot plot showing expression of cell type markers. ( E ) IGV screenshot of T1 and T2 alignments showing GNAS R844S mutation position, UMAP plot highlighting location of cells with GNAS mutation and violin plot showing relative expression of mutated and wild-type GNAS epithelial cells. ( F ) IGV screenshot of T1 and T2 alignments showing KRAS G12V mutation position, UMAP plot highlighting location of cells with KRAS mutation and violin plot showing relative expression of mutated and wild-type KRAS epithelial cells.

Article Snippet: The libraries were prepared with 900 fmol of pooled amplicon for PromethION Flow Cell FLO-PRO002 (Oxford Nanopore Technologies) using Native Barcoding Expansion and Ligation Sequencing Kit (Oxford Nanopore Technologies) as per the manufacturer’s protocol.

Techniques: Diagnostic Assay, Sequencing, Expressing, Mutagenesis

Journal: NAR Cancer

Article Title: Single-cell multi-gene identification of somatic mutations and gene rearrangements in cancer

doi: 10.1093/narcan/zcad034

Figure Lengend Snippet: Single-cell mutations from the T3 and T4 appendiceal cancers. ( A ) Location of tumor samples for patient 8629, and variants detected from clinical diagnostic sequencing having sufficient long-read depth for analysis. ( B ) IGV screenshots for T3 alignments, covering the length of KRAS and GNAS genes. ( C ) UMAP clustered plot showing integration with T3 and T4 samples. ( D ) UMAP clustered plot annotated with cell types and dot plot showing expression of cell type markers. ( E ) IGV screenshot of T3 and T4 alignments showing KRAS G12V mutation position, UMAP plot highlighting location of cells with KRAS mutation and violin plot showing relative expression of mutated and wild-type KRAS epithelial cells. ( F ) IGV screenshot of T3 and T4 alignments showing GNAS R844C and R844H mutation positions, UMAP plot highlighting location of cells with GNAS R844H mutation and violin plot showing relative expression of mutated and wild-type GNAS R844H epithelial cells.

Article Snippet: The libraries were prepared with 900 fmol of pooled amplicon for PromethION Flow Cell FLO-PRO002 (Oxford Nanopore Technologies) using Native Barcoding Expansion and Ligation Sequencing Kit (Oxford Nanopore Technologies) as per the manufacturer’s protocol.

Techniques: Diagnostic Assay, Sequencing, Expressing, Mutagenesis

Journal: NAR Cancer

Article Title: Single-cell multi-gene identification of somatic mutations and gene rearrangements in cancer

doi: 10.1093/narcan/zcad034

Figure Lengend Snippet: Single-cell mutations from the T5 and T6 B-cell lymphomas. ( A ) Location of tumor samples for patient 6408 and location of biopsy taken for clinical diagnostic sequencing, plus mutations detected from targeted sequencing having sufficient long-read depth for analysis. ( B ) IGV screenshots for T5 alignments, covering the length of CREBBP and BCL2 genes. ( C ) UMAP clustered plot showing integration of T5 and T6 samples. ( D ) UMAP clustered plot annotated with cell types and dot plot showing expression of cell type markers. ( E ) IGV screenshot of T5 and T6 alignments showing BCL2 mutation positions, UMAP plot highlighting location of cells with BCL2 S116F mutation and violin plot showing relative expression of mutated and wild-type BCL2 S116F B cells, with an asterisk indicating significant difference in expression (adjusted P -value <0.05). ( F ) IGV screenshot of T5 and T6 alignments showing CREBBP Y1482H mutation position, UMAP plot highlighting location of cells with CREBBP mutation and violin plot showing relative expression of mutated and wild-type CREBBP B cells.

Article Snippet: The libraries were prepared with 900 fmol of pooled amplicon for PromethION Flow Cell FLO-PRO002 (Oxford Nanopore Technologies) using Native Barcoding Expansion and Ligation Sequencing Kit (Oxford Nanopore Technologies) as per the manufacturer’s protocol.

Techniques: Diagnostic Assay, Sequencing, Expressing, Mutagenesis

Journal: NAR Cancer

Article Title: Single-cell multi-gene identification of somatic mutations and gene rearrangements in cancer

doi: 10.1093/narcan/zcad034

Figure Lengend Snippet: ( A ) IGV screenshots of T5 and T6 lymphomas and locations of BCL2 variants called by Longshot. Coding mutations are labeled in gray and additional variants detected by Longshot are labeled in blue. ( B ) Schematic of translocation detected by cuteSV. An IGV screenshot showing primary alignments to IGH-D2 on chromosome 14 with soft-clipped sequence to the right, plus secondary alignments of the same reads to a region downstream of BCL2 3′ UTR on chromosome 18.

Article Snippet: The libraries were prepared with 900 fmol of pooled amplicon for PromethION Flow Cell FLO-PRO002 (Oxford Nanopore Technologies) using Native Barcoding Expansion and Ligation Sequencing Kit (Oxford Nanopore Technologies) as per the manufacturer’s protocol.

Techniques: Labeling, Translocation Assay, Sequencing