Single cells were thawed briefly, followed by addition of 6 µL Single Cell Lysis Buffer (Single Cell Lysis Kit, ThermoFisher) and room temperature incubation for 1 h. A volume of 1 µL Single Cell Stop Solution (Single Cell Lysis Kit, ThermoFisher) was added, followed by room temperature incubation for 10 min. Single-cell lysate was used as template for the Ion Torrent AmpliSeq HD Pan-Cancer Single Cell Library Prep kit.
Single cell lysis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Single Cell Lysis Kit is a laboratory equipment designed to effectively lyse individual cells, thereby releasing their cellular contents for further analysis. The kit provides a standardized and efficient method for sample preparation in single-cell studies.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Puregene core kit a dna extraction kit, by Qiagen (2 mentions)
Low protein binding tube, by Eppendorf (2 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (2 mentions)
Taqman preamp master mix, by Thermo Fisher Scientific (2 mentions)
Quantstudio 6 flex real time pcr, by Thermo Fisher Scientific (2 mentions)
Capsuret macro lcm caps, by Thermo Fisher Scientific (2 mentions)
Ncounter single cell gene expression assay, by NanoString (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Anti cd19 fitc, by BD (2 mentions)
Facsaria 2 cell sorter, by BD (2 mentions)
Fc blocking reagent, by Miltenyi Biotec (2 mentions)
Agilent 2100 high sensitivity dna assay kit, by Agilent Technologies (1 mentions)
Novaseq s2, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Superscript 3 rt, by Thermo Fisher Scientific (1 mentions)
Rnaseout recombinant rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Dntp mix, by Thermo Fisher Scientific (1 mentions)
C1000tm thermal cycler, by Bio-Rad (1 mentions)
16 protocols using single cell lysis kit
1
Single-cell Transcriptome Profiling
2
DNA Extraction and Amplification from Leukemic and Hematopoietic Cells
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For bulk leukemic blasts with cell number yield >20,000 cells, gDNA was extracted using the Puregene Core kit A DNA extraction kit (Qiagen, Maryland, USA) according to manufacturer instructions. After quantification, 10 ng of gDNA was used for amplification step.
For sorted HSC, LSC, non-LSC compartments with cell number yield of 300–500 cells and single colonies collected from methylcellulose medium, cells were lysed immediately post-collection by using the Single Cell Lysis Kit (Invitrogen) as described before [20 (link)]. Briefly, 9 µL of single cell lysis solution was directly added to the droplet of sorted cells or collected colony. After 5 min incubation at room temperature, 1 µL of stop solution was added. The reaction mixture was used for amplification.
For sorted HSC, LSC, non-LSC compartments with cell number yield of 300–500 cells and single colonies collected from methylcellulose medium, cells were lysed immediately post-collection by using the Single Cell Lysis Kit (Invitrogen) as described before [20 (link)]. Briefly, 9 µL of single cell lysis solution was directly added to the droplet of sorted cells or collected colony. After 5 min incubation at room temperature, 1 µL of stop solution was added. The reaction mixture was used for amplification.
3
Single-cell RNA-seq of cloned embryos
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RNA was extracted from the 8-cell embryos fertilized and cloned using UC-MSC and fibroblasts (the three groups were labelled IVF, DFUC and DFF, respectively), and embryos were lysed using a single-cell lysis kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Subsequently, the 1st cDNA was obtained using a single-cell reverse transcription kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual and then amplified using PCR with the 1st cDNA as a template. The cDNA product was purified using Ampure XP beads and its concentration was measured with a Qubit® 3.0 Fluorometer (Thermo, Waltham, MA, USA). The integrity and distribution of fragments were assessed using an Agilent 2100 Bioanalyzer and an Agilent 2100 High Sensitivity DNA Assay Kit (Agilent, Santa Clara, CA, USA). Library construction was performed by taking 40 ng of product from each sample of satisfactory quality and breaking it into 350 bp fragments using ultrasound. The fragments then underwent end repair, addition of base A, addition of an adapter, and PCR amplification. Finally, Novaseq S2 (Illumina, San Diego, CA, USA) was used for paired-end sequencing.
4
Extraction and Amplification of Genomic DNA from Leukemic Blasts and Sorted Hematopoietic Cells
5
Single-Cell RNA Extraction and Reverse Transcription
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Each single CTC was transferred to 0.2 ml PCR tube and subjected to lysis and RNA extraction according to the manufacturer’s specifications (Single Cell Lysis Kit, Thermo Fisher Scientific, MA, USA). 2.5 μM oligo (dT) primers and 0.5 mM dNTP Mix (all Life Technologies, Singapore) were added into the lysed CTC sample, which was subsequently incubated at 65°C for 5 min and cooled on ice for at least 1 min. 1x first-strand buffer, 5 mM DTT, 10 U RNaseOUT Recombinant RNase Inhibitor, and 50 U SuperScript III RT (all Life Technologies, Singapore) were used, made up to a final volume of 20 μl in nuclease-free water. The final product was incubated at 25°C for 5 min, 55°C for 60 min, and 85°C for 5 min for reverse transcription on a C1000TM Thermal Cycler (Bio-Rad, Hercules, USA).
6
Circulating Tumor Cell Isolation and Analysis
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CD45-negative enriched cells were stained with antibodies targeting human CD45, pan-cytokeratin, and HER2/vimentin/DUSP6 prior to CTC enumeration and sorting by DEPArray V2 or NXT (Silicon Biosystems, Italy). CTCs were defined as nucleated cells lacking CD45 and expressing pan-cytokeratin (CK7, CK8, CK18, and CK19) as described previously [5 (link)]. CTCs for mRNA analysis were lysed using the Single Cell Lysis Kit (Thermo Fisher Scientific, Waltham, MA, USA) and stored at –80 °C prior to further analysis. CTCs for immunofluorescence microscopy analysis were cytospun onto coverslips and fixed prior to fluorescence labeling and imaging.
7
Single-Cell Gene Expression of hiPSC-CMs
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Single cells were collected using a single use glass pipette of a Scanning Ion Conductance Microscope. Negative pressure was applied to create a small suction to remove a single isolated hiPSC-CM from the platform. The sample was then collected in a low protein binding tube (Eppendorf). Total RNA was isolated using the Single Cell Lysis Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol, and cDNA produced using SuperScript® VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific), assuming a 1:1 conversion. cDNA was pre-amplified with 0.2X Taqman® probes, see below, and TaqMan® PreAmp Master Mix (Thermo Fisher Scientific) and diluted with TE buffer to 133 µl. Single cell Quantitative PCR was performed using 4 µl of cDNA with TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific) and QuantStudio™ 6 Flex Real-Time PCR (Thermo Fisher Scientific) and the following TaqMan® probes: GAPDH (Hs02758991_g1), MYL2 (Hs00166405_m1), MYL7 (Hs01085598_g1), MYH6 (Hs01101425_m1), MYH7 (Hs01110632_m1), TNNI1 (Hs00913333_m1) and TNNI3 (Hs00165957_m1). The ΔΔCt method was used to compare expression between single hiPSC-CMs cultured on 3D micropatterned substrates and flat control surfaces and normalized to hiPSC-CMs before plating (N = 4, n ⩾ 10).
8
Single Cell qPCR of hiPSC-CMs
9
Laser Capture Micro-dissection and Single-Cell Expression Analysis
10
Laser Capture Micro-dissection and Single-Cell Expression Analysis
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Arcturus XT (Life Technologies, Carlsbad, CA, USA) was used to perform laser capture microdissection. Cells showing lentiviral vector infection were captured by hand and transferred onto a cap (Capsuret Macro LCM Caps, Life Technologies, catalog number LCM0211) using a near-infrared laser pulse. RNA was extracted from caps using Single-Cell Lysis Kit following the manufacturer’s instruction (Life Technologies, Grand Island, NY, USA). Given the very low amount of RNA collected, we measured mRNA expression using the nCounter Single-Cell Gene Expression assay following the manufacturer’s instruction (Nanostring Technologies, Seattle, WA, USA).
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