The GE Healthcare illustra GenomiPhi V2 DNA Amplification Kit and the Qiagen REPLI-g single cell kit were used on both tube and microfluidics platform. For the Qiagen REPLI-g single cell kit, the protocol recommended 8 h of incubation time.
Repli g single cell kit
Manufactured by Qiagen
Sourced in Germany, United States, Australia, United Kingdom, Netherlands
The REPLI-g Single Cell Kit is a laboratory product designed for whole genome amplification (WGA) from single cells. It enables the generation of microgram quantities of DNA from a single cell, suitable for downstream genomic analysis applications.
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230 protocols using repli g single cell kit
1
Comprehensive DNA Amplification Protocols
2
Single-Cell Copy Number Variation Analysis
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Example 11
In this example, the method provided herein is compared with some current single cell preparation methods.
3
Single-cell isolation and WGA of phagocytes and sperm
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SpTrf+ small phagocytes and red spherule cells were sorted as single cells into a 384-well plate containing 7 μl CMFSW-EH per well and observed by light and/or fluorescence microscopy to verify the presence of a single cell of the expected type in a well. After the verification of cell type per well, three SpTrf+ and three red spherule cells from each animal before challenge on day 0 and on days 1 and 2 after challenge were evaluated further. Cells were sorted into 4 μl D2 lysis buffer plus 3 μl 3.3X PBS of the REPLI-g Single Cell kit (Qiagen) and stored at −80°C until processing for WGA.
Sperm were collected from sea urchins after electric shock with a current of 16–20 mA for 1–2 min to induce spawning. Sperm (106) cells were diluted into 1 ml of CMFSW-EH and stained with Hoechst 33342 (5 μg/ml) and PI (1 μg/ml) to determine the live/dead cell ratio. Stained cells were washed with the CMFSW-EH and diluted to a concentration of 0.25 cells/μl. Aliquots of 4 μl were placed in wells of a 384-well plate to achieve a statistical concentration of 0.1 cell per well. Wells with single Hoechst 33342+/PI− sperm were identified by fluorescence microscopy and 4 μl D2 lysis buffer of the REPLI-g Single Cell kit (Qiagen) was added to each followed by WGA as described below.
Sperm were collected from sea urchins after electric shock with a current of 16–20 mA for 1–2 min to induce spawning. Sperm (106) cells were diluted into 1 ml of CMFSW-EH and stained with Hoechst 33342 (5 μg/ml) and PI (1 μg/ml) to determine the live/dead cell ratio. Stained cells were washed with the CMFSW-EH and diluted to a concentration of 0.25 cells/μl. Aliquots of 4 μl were placed in wells of a 384-well plate to achieve a statistical concentration of 0.1 cell per well. Wells with single Hoechst 33342+/PI− sperm were identified by fluorescence microscopy and 4 μl D2 lysis buffer of the REPLI-g Single Cell kit (Qiagen) was added to each followed by WGA as described below.
4
Single-Cell Copy Number Variation Analysis
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Example 11
In this example, the method provided herein is compared with some current single cell preparation methods.
5
Single-Cell Whole Genome Amplification via Droplets
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A QX200 Droplet Generator (BIO-RAD, CA, USA) in combination with Pico-Surf 1 (2% in Novec 7500) oil (Dolomite Bio, UK) was used for droplet generation. MDA was performed according to protocol 1: Amplification of genomic DNA from single cells from the REPLI-g Single Cell Kit (Qiagen, MD, USA), with minor modifications to be compatible with the droplet generator; Since the standard sample input to the Droplet Generator is 20 μl, each sonicated cell/DNA sample (which contained 15 μl) was separated into two equal samples and each mixed with components from the REPLI-g Single Cell Kit to a total of 21 μl. 20 μl of this reaction was loaded on to the Droplet Generator and mixed with 40 μl Picro-Surf 1 (2% in Novec 7500) oil. After droplet generation, the amplification was run at 30 °C for eight hours. The emulsion was then broken and the single amplified genomes (SAGs) were recovered following the “Amplicon Recovery from Droplets” protocol (BIO-RAD).
6
Single-Cell Yeast Genome Amplification
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Eighty wells of a 96-well PCR plate were each filled with 1 μl of PBS buffer (pH 8.0) before single-cell droplet collection. Sixty droplets each with a single S. cerevisiae cell and twenty blank droplets were then collected successively in these wells. Cells were recovered from the droplets as described above and were lysed by adding 1.5 μl of buffer D2 (REPLI-g Single Cell Kit; Qiagen, USA) containing 0.08 mol/L dithiothreitol (DTT) and incubation at 65 oC for 10 minutes, followed by neutralization with 1.5 μl of Stop Solution (REPLI-g Single Cell Kit; Qiagen, USA). The volume of droplet after cell lysis in each of the wells was about 5 μl. A 162-bp fragment of yeast ALG9 gene was then amplified using the cell lysates as template (Supplementary Table S1 )45 (link). The remaining sixteen wells on the same plate were used for standard curve measurement using DNA extracted from yeast bulk culture. Each 20-μl reaction was performed and monitored on a LightCycler 480 Real-Time PCR System (Roche Applied Science, USA). Experimental details were provided in the Supplementary Information .
7
Single-Cell Whole Genome Amplification
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WGA Reactions were assembled in a pre-PCR workstation that provides constant positive pressure with HEPA filtered air and which was decontaminated with UV light for 30 minutes before each experiment. MDA was carried according to the SCMDA methodology using the REPLI-g Single Cell Kit (Qiagen) according the published protocol (16) . PTA was carried out by first further lysing the cells after freeze thawing by adding 2 µL a prechilled solution of a 1:1 mixture of 5% Triton X-100 (Sigma-Aldrich) and 20 mg/ml Proteinase K (Promega). The cells were then vortexed and briefly centrifuged before placing at 40ºC for 10 minutes. 4 µL of Buffer D2 (REPLI-g Single Cell Kit, Qiagen) and 1 µL of 500 µM exonuclease-resistant random primer were then
8
Myeloid Progenitor Cell Isolation
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1 ml viably frozen bone marrow MNCs were thawed in the presence of 100 μl DNAse I (2 mg μl−1) and incubated for 10 min in a solution of 1.6 ml fetal calf serum, 10 μl heparin (5,000 U ml−1) and 100 μl MgSO4 (0.22 μM). Subsequently, the myeloid progenitor cells were sorted according to a protocol adapted from Pang et al.35 (link) The cells were washed and stained with CD34-APC (Beckman Coulter, Brea, CA, USA), CD38-PE-Cy7 (BioLegend, San Diego, CA, USA), CD123-PE (BioLegend) and CD45RA-PB (BioLegend) monoclonal antibodies. Cells were analysed and sorted using a FACS Aria SORP flow cytometer and DIVA software (Becton Dickinson, Franklin Lakes, NJ, USA). Viable cells were selected based on forward scatter and side scatter profiles, and doublets were discriminated using forward scatter area versus width and side scatter area versus width. The HSC population was defined as CD34+CD38−. Within the CD34+CD38+ fraction, the common myeloid progenitor cells (CD123+CD45RA−), the granulocyte-macrophage progenitor cells (CD123+CD45RA+) and the megakaryocyte-erythroid progenitor cells (CD123−CD45RA−) were selected. DNA isolation from these cell fractions, followed by DNA amplification, was carried out using the Qiagen REPLI-g single cell kit (Qiagen) according to the manufacturer’s protocol.
9
Whole Genome Amplification for Metagenomic Sequencing
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The total DNA of sample 1.2 and 7.2 were isolated and amplified by QIAGEN REPLI-g Single Cell Kit (QIAGEN, American) according to the kit operation manual to get whole genome amplified. Only 10–15 ng total DNA was used as the initial template for the WGA, and then the product was used as the template of the following PCR, gel recovery and Miseq sequencing for sample 1.2 and 7.2.
10
Single Cell Whole Genome Amplification and SNP Genotyping
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MDA was performed using REPLI-g Single Cell Kit (150,345, QIAGEN, Germany) in SNP array according to the authors’ previous report [19 (link)]. Briefly, biopsied TE cells (5 to 10 cells) were lysed in DTT and DLB buffer. Whole genome DNA was amplified in an amplification buffer created by mixing REPLI-g single-cell DNA polymerase and the reaction buffer. The MDA product was then hybridized on the Illumina HumanCyto12 microarray by following the user manual. Data were analyzed using GenomeStudio Software (2011, Illumina, USA). The genotype was evaluated by using the B allele frequency and log2R ratio (log2R = Log2 normalized R value/expected normalized R value).
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