Journal: Current protocols in molecular biology

Article Title: The use of the Fluidigm C1 for RNA expression analyses of single cells

doi: 10.1002/cpmb.55

Figure Lengend Snippet: Overview of using the Fluidigm C1 system to capture single-cells and generate cDNA libraries for next-generation sequencing as described in (A) Basic Protocol 1 and (B) Basic Protocol 2. Sequences are not drawn to scale.

Article Snippet: C1 Single-Cell Reagent Kit for mRNA Seq (Fludigm Catalogue number: 100–6201) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 C1 Preloading Reagent (purple lid) C1 Blocking Reagent (white lid) Loading Reagent (yellow lid) Suspension Reagent (green lid) C1 Harvest Reagent SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (Takara, Catalogue number: 634833) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 RNase Inhibitor 3’SMART CDS Primer IIA SMARTer Dilution Buffer 5x First-Strand Buffer (RNase-Free) Dithioreitol (DTT) dNTP Mix SMARTer IIA oligonucleotide SMARTScribe Reverse Transcriptase Advantage 2 PCR Kit (Takara, Catalogue number: 639206) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 50x Advantage 2 Polymerase Mix 10X Advantage 2 PCR Buffer 50X dNTP Mix IS PCR Primer PCR-Grade Water C1 IFC for mRNA seq (5–10 μm, 10–17 μm, or 17–25 μm) (Fluidigm) in 3 capture site sizes: list-behavior=simple prefix-word= mark-type=none max-label-size=0 5–10 μM (Catalogue number: 100–5759) 10–17 μM (Catalogue number: 100–5760) 17–25 μM (Catalogue number: 100–5761) C1 System (Fluidigm, Catalogue number: 100–7000) list-behavior=simple prefix-word= mark-type=none max-label-size=0 0.2 mL PCR Tubes, Nuclease-Free 1.5 mL Eppendorf Tubes, Nuclease-Free Agilent 2100 Bioanalyzer Hot-lid thermal cycler PCR Hood 8 Channel Pipette Magnetic Stand for 1.5 mL Eppendorf Tubes Centrifuge for 1.5-mL tubes Centrifuge for 96-well plates Prepare Reagents for C1 operation list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Thaw reagents Thaw on ice: RNase inhibitor, 3’SMART CDS Primer IIA, 5X First-Strand Buffer, Dithiothreitol, dNTP Mix, SMARTScribe Reverse Transcriptase, 10X Advantage 2 PCR Buffer, 50X dNTP Mix, IS PCR Primer, 50X Advantage 2 Polymerase Mix Equilibrate to room temperature: C1 Preloading Reagent, C1 Harvest Reagent, C1 Blocking Reagent, Loading Reagent, SMARTer dilution buffer, SMARTer IIA Oligonucleotide, Suspension Reagent, C1 DNA Dilution Reagent Note: It is important to minimize the time enzymes are not at normal storage temperature to preserve activity.

Techniques: Next-Generation Sequencing

Journal: Current protocols in molecular biology

Article Title: The use of the Fluidigm C1 for RNA expression analyses of single cells

doi: 10.1002/cpmb.55

Figure Lengend Snippet: Loading guide for Fluidigm C1

Article Snippet: C1 Single-Cell Reagent Kit for mRNA Seq (Fludigm Catalogue number: 100–6201) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 C1 Preloading Reagent (purple lid) C1 Blocking Reagent (white lid) Loading Reagent (yellow lid) Suspension Reagent (green lid) C1 Harvest Reagent SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (Takara, Catalogue number: 634833) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 RNase Inhibitor 3’SMART CDS Primer IIA SMARTer Dilution Buffer 5x First-Strand Buffer (RNase-Free) Dithioreitol (DTT) dNTP Mix SMARTer IIA oligonucleotide SMARTScribe Reverse Transcriptase Advantage 2 PCR Kit (Takara, Catalogue number: 639206) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 50x Advantage 2 Polymerase Mix 10X Advantage 2 PCR Buffer 50X dNTP Mix IS PCR Primer PCR-Grade Water C1 IFC for mRNA seq (5–10 μm, 10–17 μm, or 17–25 μm) (Fluidigm) in 3 capture site sizes: list-behavior=simple prefix-word= mark-type=none max-label-size=0 5–10 μM (Catalogue number: 100–5759) 10–17 μM (Catalogue number: 100–5760) 17–25 μM (Catalogue number: 100–5761) C1 System (Fluidigm, Catalogue number: 100–7000) list-behavior=simple prefix-word= mark-type=none max-label-size=0 0.2 mL PCR Tubes, Nuclease-Free 1.5 mL Eppendorf Tubes, Nuclease-Free Agilent 2100 Bioanalyzer Hot-lid thermal cycler PCR Hood 8 Channel Pipette Magnetic Stand for 1.5 mL Eppendorf Tubes Centrifuge for 1.5-mL tubes Centrifuge for 96-well plates Prepare Reagents for C1 operation list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Thaw reagents Thaw on ice: RNase inhibitor, 3’SMART CDS Primer IIA, 5X First-Strand Buffer, Dithiothreitol, dNTP Mix, SMARTScribe Reverse Transcriptase, 10X Advantage 2 PCR Buffer, 50X dNTP Mix, IS PCR Primer, 50X Advantage 2 Polymerase Mix Equilibrate to room temperature: C1 Preloading Reagent, C1 Harvest Reagent, C1 Blocking Reagent, Loading Reagent, SMARTer dilution buffer, SMARTer IIA Oligonucleotide, Suspension Reagent, C1 DNA Dilution Reagent Note: It is important to minimize the time enzymes are not at normal storage temperature to preserve activity.

Techniques:

Journal: Current protocols in molecular biology

Article Title: The use of the Fluidigm C1 for RNA expression analyses of single cells

doi: 10.1002/cpmb.55

Figure Lengend Snippet: Sample harvest guide for Fluidigm C1

Article Snippet: C1 Single-Cell Reagent Kit for mRNA Seq (Fludigm Catalogue number: 100–6201) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 C1 Preloading Reagent (purple lid) C1 Blocking Reagent (white lid) Loading Reagent (yellow lid) Suspension Reagent (green lid) C1 Harvest Reagent SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (Takara, Catalogue number: 634833) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 RNase Inhibitor 3’SMART CDS Primer IIA SMARTer Dilution Buffer 5x First-Strand Buffer (RNase-Free) Dithioreitol (DTT) dNTP Mix SMARTer IIA oligonucleotide SMARTScribe Reverse Transcriptase Advantage 2 PCR Kit (Takara, Catalogue number: 639206) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 50x Advantage 2 Polymerase Mix 10X Advantage 2 PCR Buffer 50X dNTP Mix IS PCR Primer PCR-Grade Water C1 IFC for mRNA seq (5–10 μm, 10–17 μm, or 17–25 μm) (Fluidigm) in 3 capture site sizes: list-behavior=simple prefix-word= mark-type=none max-label-size=0 5–10 μM (Catalogue number: 100–5759) 10–17 μM (Catalogue number: 100–5760) 17–25 μM (Catalogue number: 100–5761) C1 System (Fluidigm, Catalogue number: 100–7000) list-behavior=simple prefix-word= mark-type=none max-label-size=0 0.2 mL PCR Tubes, Nuclease-Free 1.5 mL Eppendorf Tubes, Nuclease-Free Agilent 2100 Bioanalyzer Hot-lid thermal cycler PCR Hood 8 Channel Pipette Magnetic Stand for 1.5 mL Eppendorf Tubes Centrifuge for 1.5-mL tubes Centrifuge for 96-well plates Prepare Reagents for C1 operation list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Thaw reagents Thaw on ice: RNase inhibitor, 3’SMART CDS Primer IIA, 5X First-Strand Buffer, Dithiothreitol, dNTP Mix, SMARTScribe Reverse Transcriptase, 10X Advantage 2 PCR Buffer, 50X dNTP Mix, IS PCR Primer, 50X Advantage 2 Polymerase Mix Equilibrate to room temperature: C1 Preloading Reagent, C1 Harvest Reagent, C1 Blocking Reagent, Loading Reagent, SMARTer dilution buffer, SMARTer IIA Oligonucleotide, Suspension Reagent, C1 DNA Dilution Reagent Note: It is important to minimize the time enzymes are not at normal storage temperature to preserve activity.

Techniques:

Journal: Current protocols in molecular biology

Article Title: The use of the Fluidigm C1 for RNA expression analyses of single cells

doi: 10.1002/cpmb.55

Figure Lengend Snippet: Trouble Shooting

Article Snippet: C1 Single-Cell Reagent Kit for mRNA Seq (Fludigm Catalogue number: 100–6201) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 C1 Preloading Reagent (purple lid) C1 Blocking Reagent (white lid) Loading Reagent (yellow lid) Suspension Reagent (green lid) C1 Harvest Reagent SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (Takara, Catalogue number: 634833) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 RNase Inhibitor 3’SMART CDS Primer IIA SMARTer Dilution Buffer 5x First-Strand Buffer (RNase-Free) Dithioreitol (DTT) dNTP Mix SMARTer IIA oligonucleotide SMARTScribe Reverse Transcriptase Advantage 2 PCR Kit (Takara, Catalogue number: 639206) Contains: list-behavior=simple prefix-word= mark-type=none max-label-size=0 50x Advantage 2 Polymerase Mix 10X Advantage 2 PCR Buffer 50X dNTP Mix IS PCR Primer PCR-Grade Water C1 IFC for mRNA seq (5–10 μm, 10–17 μm, or 17–25 μm) (Fluidigm) in 3 capture site sizes: list-behavior=simple prefix-word= mark-type=none max-label-size=0 5–10 μM (Catalogue number: 100–5759) 10–17 μM (Catalogue number: 100–5760) 17–25 μM (Catalogue number: 100–5761) C1 System (Fluidigm, Catalogue number: 100–7000) list-behavior=simple prefix-word= mark-type=none max-label-size=0 0.2 mL PCR Tubes, Nuclease-Free 1.5 mL Eppendorf Tubes, Nuclease-Free Agilent 2100 Bioanalyzer Hot-lid thermal cycler PCR Hood 8 Channel Pipette Magnetic Stand for 1.5 mL Eppendorf Tubes Centrifuge for 1.5-mL tubes Centrifuge for 96-well plates Prepare Reagents for C1 operation list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Thaw reagents Thaw on ice: RNase inhibitor, 3’SMART CDS Primer IIA, 5X First-Strand Buffer, Dithiothreitol, dNTP Mix, SMARTScribe Reverse Transcriptase, 10X Advantage 2 PCR Buffer, 50X dNTP Mix, IS PCR Primer, 50X Advantage 2 Polymerase Mix Equilibrate to room temperature: C1 Preloading Reagent, C1 Harvest Reagent, C1 Blocking Reagent, Loading Reagent, SMARTer dilution buffer, SMARTer IIA Oligonucleotide, Suspension Reagent, C1 DNA Dilution Reagent Note: It is important to minimize the time enzymes are not at normal storage temperature to preserve activity.

Techniques: Concentration Assay, Cell Isolation, Amplification, Isolation

Journal: PeerJ

Article Title: Mosquitoes on a chip—environmental DNA-based detection of invasive mosquito species using high-throughput real-time PCR

doi: 10.7717/peerj.17782

Figure Lengend Snippet: eDNA samples of Ae. japonicus and Ae. koreicus sites were collected at multiple random open water basins at four cemeteries in Hessen with three different filter media with varying pore sizes, whereas Ae. albopictus sites were sampled at divers water reservoirs and breeding water solely with Sterivex ® filters until clogging. After eDNA extraction the samples where analyzed via Real-Time PCR and high-throughput real-time PCR on a Biomark HD platform with 96.96 Dynamic IFCs. Created with BioRender.com with mosquito illustrations from J. L. Ordóñez.

Article Snippet: Since the data produced by Biomark IFCs is based on fluorescence measurements similarly to single-species qPCR, data analysis and interpretation is comparably easy, fast and highly reproducible.

Techniques: Extraction, Real-time Polymerase Chain Reaction, High Throughput Screening Assay