Single-cells not cryopreserved after FACS (fresh) were assayed as previously described (Buenrostro et al., 2015b (link)). To assay cells cryopreserved after FACS (frozen), cells were allowed to recover for 10 min at 37°C in IMDM with 10% FBS. After recovery, cells were washed twice in cold 1× PBS and once in with the C1 DNA Seq Cell Wash Buffer (Fluidigm). Cells were then resuspended in 6 µl of C1 DNA Seq Cell Wash Buffer, and were combined with 4 µl of C1 Cell Suspension Reagent, 7 µl of this cell mix was loaded onto the Fluidigm IFC. Single-cells were then assayed using scATAC-seq as previously described (Buenrostro et al., 2015b (link)).
C1 dna seq cell wash buffer
Manufactured by Standard BioTools
The C1 DNA Seq Cell Wash Buffer is a laboratory reagent designed to prepare and clean cell samples for DNA sequencing applications. It is used to remove impurities and unwanted materials from the cell samples, ensuring the integrity and purity of the DNA for downstream sequencing analysis.
Automatically generated - may contain errors
Lab products found in correlation
Open app program, by Standard BioTools (2 mentions)
C1 ifc microfluidic chips, by Standard BioTools (2 mentions)
C1 script builder software, by Standard BioTools (2 mentions)
Ctr 6000 microscope, by Leica (2 mentions)
C1 single cell auto prep system, by Standard BioTools (2 mentions)
Live dead cell viability assay, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by GE Healthcare (1 mentions)
C1 chip, by Standard BioTools (1 mentions)
C1 harvest reagent, by Standard BioTools (1 mentions)
Dtt mix, by GE Healthcare (1 mentions)
Picogreen dsdna quantification assay, by Thermo Fisher Scientific (1 mentions)
Infinite 200 pro plate reader, by Tecan (1 mentions)
5 protocols using c1 dna seq cell wash buffer
1
scATAC-seq on Cryopreserved Cells
2
Single-cell RNA-sequencing of reprogramming cells
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The scRNA-Seq libraries were prepared following the published protocol with minor modifications (11 (link)). Briefly, cells undergoing reprogramming were trypsinized into single cells at each time point. Cells were washed three times with C1 DNA-seq Cell Wash Buffer (Fluidigm). Cells at a concentration of 300 to 600 cells/μl were mixed with C1 Cell Suspension Reagent at a ratio of 3:2. Next, the cell mixture was loaded into C1 Single-Cell Auto Prep IFC (integrated fluidic circuits) microfluidic chips according to the “STRT seq, 1862× (10-17 μm diameter cells)” protocol. Cells were captured at the 96 capture sites in the microfluidic chip and stained using a green fluorescent calcein-AM dye (LIVE/DEAD cell viability assay, Life Technologies) and imaged by a Leica CTR 6000 microscope. mRNA-Seq libraries were prepared following the Generate complementary DNA (cDNA) libraries with the C1 Single-Cell mRNA Seq HT IFC and Reagent Kit V2 manual.
3
Single-Cell T-ATAC-Seq Using Fluidigm C1
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We adapted the C1 Single-Cell Auto Prep System with its Open App™ program (Fluidigm, Inc.) to perform T-ATAC-seq. Single T cells were captured using the C1 IFC microfluidic chips (small; 5–10micron) and custom-built T-ATAC-seq scripts generated using the C1™ Script Builder Software (scripts available from Fluidigm and upon request). Jurkat cells or peripheral blood T cells were first isolated by FACS sorting and then washed three times in C1 DNA Seq Cell Wash Buffer (Fluidigm). Cells were resuspended in DNA Seq Cell Wash Buffer at a concentration of 300 cells/μL and mixed with C1 Cell Suspension Reagent at a ratio of 3:2. 15μL of this cell mix was loaded onto the IFC. After cell loading, captured cells were visualized by imaging on a Leica CTR 6000 microscope.
4
Single-Cell T-ATAC-Seq Using Fluidigm C1
5
Single-Cell Whole Genome Amplification
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The medium-sized (10 to 17 µm) C1 chip (Fluidigm) was primed with C1 Harvest Reagent, Preloading Reagent, Blocking Reagent and C1 DNA Seq Cell Wash Buffer (Fluidigm) for 10 min before it was loaded with the dissociated single cells. The DTT Mix was prepared by the addition of DTT, Sample and Reaction Buffers (GE Healthcare). The Lysis Mix contained C1 DNA Seq Lysis Buffer and DTT (Fluidigm), while the Reaction-Enzyme Mix consisted of C1 DNA Seq Reaction Mix (Fluidigm), DTT Mix and Enzyme Mix (GE Healthcare). The Lysis Mix, Reaction-Enzyme Mix and C1 DNA Seq Stop Buffer were loaded on the C1 chip followed by the on-chip whole genome amplification experiment. The amplified DNA was harvested from the C1 chip and transferred into 96-well PCR plate. The DNA was quantified using PicoGreen dsDNA quantification assay (Thermo Fisher) on the Infinite 200Pro plate reader (Tecan).
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