Single cells were captured on medium sized Fluidigm C1 Single Cell Integrated Fluidic Circuit (IFC). The SMARTer Ultra Low RNA Kit for Illumina (Clontech) was used for single-cell capture (verified via microscope), on-chip lysis, reverse transcription, and complementary DNA (cDNA) generation. An ERCC Spike-in Mix (Ambion) was used as the technical control per Fluidigm recommendation. Single-cell cDNA quantification was performed using Agilent High Sensitivity DNA Kits and Quant-it Picogreen dsDNA (Invitrogen). cDNA was normalized to 0.20ng/μL with Biomek NXP(Beckman Coulter). The Nextera XT DNA Library Prep Kit (Illumina) was used for dual indexing and amplification following the Fluidigm C1 protocol. Libraries were purified and size selected twice using volume of Agencourt AMPure XP beads (Beckman Coulter). Cleaned libraries were quantified with Quant-it Picogreen dsDNA (Invitrogen) and normalized to 0.3ng/µl with Biomek NXP (Beckman Coulter). Single-cell RNA sequencing libraries were subsequently pooled for 96-plex sequencing. The resulting cDNA libraries were quantified using High Sensitivity DNA Kit (Agilent). The pooled 96x single-cell libraries were sequenced using HiSeq 2500 (Illumina) with paired end 126bp-8bp-8bp-126bp and 14pM-loading concentration with 5% PhiX spike-in.
Biomek nxp
Manufactured by Beckman Coulter
Sourced in United States, Germany
The Biomek NXP is a liquid handling workstation designed for laboratory automation. It is capable of performing a wide range of liquid handling tasks, including sample transfer, serial dilution, and reagent addition. The Biomek NXP is equipped with a robotic arm and a variety of interchangeable pipetting tools to ensure precise and accurate liquid handling.
Automatically generated - may contain errors
Lab products found in correlation
Multidrop combi, by Thermo Fisher Scientific (4 mentions)
Spectramax m2e, by Molecular Devices (2 mentions)
Ercc spike in mix, by Thermo Fisher Scientific (2 mentions)
Quant it picogreen dsdna, by Thermo Fisher Scientific (2 mentions)
Nextera xt dna library prep kit, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
High sensitivity dna kit, by Agilent Technologies (2 mentions)
Epitect 96 bisulfite kit, by Qiagen (2 mentions)
Hotstartaq master mix kit, by Qiagen (2 mentions)
C1000 thermal cycler, by Bio-Rad (2 mentions)
Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (2 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Epitect bisulfite kit, by Qiagen (1 mentions)
Dnadvance genomic dna isolation kit, by Beckman Coulter (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Nippon Gene (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Oxidized ldl elisa, by Mercodia (1 mentions)
Chemagic msm 1 system, by PerkinElmer (1 mentions)
32 protocols using biomek nxp
1
Single-Cell RNA Sequencing with Fluidigm C1
2
DNA and RNA Purification from HepG2 Cells
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Total DNA was purified from REP-HepG2-NTCP cells and naive HepG2-NTCP-C4 cells using an Agencourt DNAdvance Genomic DNA Isolation kit and a Biomek NXp automatic dispenser from Beckman Coulter according to the manufacturer’s instructions. Total RNA was purified using an Agencourt RNAdvance Cell v2 and a Biomek NXp automatic from Beckman Coulter according to the manufacturer’s instructions. Before elution, the RNA samples were treated with five units DNase I (Nippon Gene) at 25°C for 15 min. For the microarray analysis, total RNA was purified from HepG2 cells using a miRNeasy Mini kit (Qiagen) according to the manufacturer’s recommendations, and samples were treated with 1 mL of DNase at 37°C for 30 min using a TURBO DNA-free kit (Ambion). DNA and RNA sample concentrations were determined using a DeNovix spectrophotometer.
3
Single-Cell RNA Sequencing with Fluidigm C1
4
ABCB1 Transporter Inhibition Assay
5
DNA Extraction and Bisulfite Conversion
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Genomic DNA from whole EDTA blood was extracted by means of the QIAamp® DNA Blood Mini Kit (QIAGEN AG, Hilden, Germany), according to the manufacturer’s protocol on a Biomek Nxp (Beckman-Coulter, Krefeld, Germany). For the analysis of methylation rates, 500 ng genomic DNA from every sample was bisulfite-converted using the EpiTect® Bisulfite Kit (QIAGEN AG, Hilden, Germany). Using this procedure, unmethylated cytosines are converted into uracils via deamination (after polymerase chain reaction (PCR): thymins), whereas methylated cytosines are protected from alteration36 (link),37 (link).
6
Bisulfite Sequencing of DNA Methylation
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DNA was bisulfite-converted using the Epitect conversion kit (Qiagen, Hilden, Germany) according to manufacturer recommendations.
Bisulfite-converted DNA was used for PCR amplification using specific primer sets (see Additional file1 : Table S1) in a Touchdown PCR approach [50 (link)]. Resulting amplicons were subjected to linear sequencing PCR using BigDye Terminator according to manufacturer instructions (ABI Life Technologies, Grand Island, USA). For Sequence cleanup prior to sequencing we used AMPure beads on a Biomek NxP liquid handling platform (Beckman Coulter, Brea, USA). Purified reactions were sequenced using a 3500xl 24 capillary Sequencer (ABI Life Technologies, Grand Island, USA).
CpG position is provided in relation to the transcriptional start site located at GRCh38:7:128241278 according to ENSEMBL gene accession # ENSG00000174697. All reported locations are in the proximal promoter upstream of the gene locus. Sequence analysis and determination of methylation rates for each CpG site were conducted using the Epigenetic Sequencing Methylation analysis software [51 (link)]. The methylation rate of each CpG site per subject was estimated by determining the ratio between normalized peak values of cytosine and thymine.
Bisulfite-converted DNA was used for PCR amplification using specific primer sets (see Additional file
CpG position is provided in relation to the transcriptional start site located at GRCh38:7:128241278 according to ENSEMBL gene accession # ENSG00000174697. All reported locations are in the proximal promoter upstream of the gene locus. Sequence analysis and determination of methylation rates for each CpG site were conducted using the Epigenetic Sequencing Methylation analysis software [51 (link)]. The methylation rate of each CpG site per subject was estimated by determining the ratio between normalized peak values of cytosine and thymine.
7
Gnotobiotic Microbiome Inoculation in Flies
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We identified five unique species in our laboratory flies (Lp, Lb, Ap, At, and Ao) (17 (link)), which we then isolated in culture. To prepare the inoculum for flies, bacteria were grown overnight in De Man, Rogosa, and Sharpe (MRS) medium in a 30 °C Innova 4000 shaker (New Brunswick) at 200 rpm. The bacteria were resuspended at 108 cells per milliliter in sterile PBS for fly gnotobiotic preparations (20 (link)). A total of 5 × 106 CFUs (50 μL of 108 bacteria per milliliter in 1× PBS) were inoculated per fly vial. The 32 combinations of the five bacterial strains were mixed using a Beckman Coulter Biomek NXP workstation to standardize the inoculum. Germ-free mated flies 5–7 d posteclosion were sorted into these vials.
8
High-Throughput Screening of PAD2 Inhibitors
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PAD2
Screening Buffer (8 μL; column
1) and PAD2 (8 μL; 2 μM final) in Screening Buffer (columns
2–23) were added to a black 384-well microtiter plate (Greiner
784076) using the FRD. Controls (column 1: DMSO (no PAD2, high control);
column 2: 5 mM Cl-amidine (high control); and column 23: DMSO (no
inhibitors, low control)) and LOPAC molecules (columns 3–22)
were pinned 2× using the 10 nL head on a Beckman Coulter BioMek
NXP to achieve a final concentration of 11 μM. After a 20 min
incubation, RFA in Screening Buffer (2 μL; 75 nM final) was
added using the FRD. The plates were read after incubating for 6 h
at 37 °C as described above.
Screening Buffer (8 μL; column
1) and PAD2 (8 μL; 2 μM final) in Screening Buffer (columns
2–23) were added to a black 384-well microtiter plate (Greiner
784076) using the FRD. Controls (column 1: DMSO (no PAD2, high control);
column 2: 5 mM Cl-amidine (high control); and column 23: DMSO (no
inhibitors, low control)) and LOPAC molecules (columns 3–22)
were pinned 2× using the 10 nL head on a Beckman Coulter BioMek
NXP to achieve a final concentration of 11 μM. After a 20 min
incubation, RFA in Screening Buffer (2 μL; 75 nM final) was
added using the FRD. The plates were read after incubating for 6 h
at 37 °C as described above.
9
Cardiometabolic Risk Factors Assessment
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Height was measured with a Harpenden stadiometer, weight recorded with a balance beam or electronic scale, and BMI calculated as weight (kg) divided by height squared (m2) at the MrOS Sleep baseline visit. Participants self‐reported if they were a current or a past smoker, if they were currently taking aspirin or a statin medication, whether or not they were using medications for hypertension, and whether or not they had been diagnosed previously by a physician with a myocardial infarction, stroke, atrial fibrillation, congestive heart failure, chronic obstructive pulmonary disease, diabetes mellitus, Parkinson's disease, rheumatoid arthritis, liver disease, or renal disease. Systolic blood pressure was measured in the right arm twice with the participant sitting, and averaged. Oxidized low‐density lipoproteins were measured on Beckman Coulter Biomek NXp (Beckman Coulter, Inc, Fullerton, CA) using a direct sandwich enzyme immunoassay (Oxidized LDL ELISA; Mercodia AB, Uppsala, Sweden).
10
DNA Methylation Analysis from Blood
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DNA for methylation analysis of both cohorts was extracted from blood. The cleanup of genomic DNA from healthy participants was performed using the Nucleo-Mag® Blood 200 μl DNA Kit (Macherey-Nagel, Düren, Germany). Genomic DNA from Crohn patients was extracted according to standard procedures with an automated chemagic MSM I system (Perkin Elmer, Baesweiler, Germany). Bisulfite conversion and DNA purification were conducted via the EpiTect® 96 Bisulfite Kit (QIAGEN, Hilden, Germany). DNA concentrations were determined via a Nanodrop 1000 spectrophotometer (VWR, Radnor, PA, USA). A Biomek® NxP (Beckman Coulter, Brea, CA, USA) was used for pipetting, transferring and purification steps.
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