ScRNA-seq libraries were generated using the 10x Chromium Controller and the Chromium Single Cell 5′ Library Construction Kit and human B cell and T cell V(D)J enrichment kit according to the manufacturer’s instructions. Briefly, the suspended cells were loaded on a Chromium controller Single-Cell Instrument to generate single-cell Gel Bead-In-Emulsions (GEMs) followed by reverse transcription and sample indexing using a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (BioRad). After breaking the GEMs, the barcoded cDNA was purified and amplified, followed by fragmenting, A-tailing and ligation with adaptors. Finally, PCR amplification was performed to enable sample indexing and enrichment of scRNA-Seq libraries. For T cell and B cell receptor sequencing, target enrichment from cDNA was conducted according to the manufacturer’s instructions. The final libraries were quantified using a Qubit dsDNA HS Assay kit (Invitrogen) and a High Sensitivity DNA chip run on a Bioanalyzer 2100 system (Agilent).
96 deep well reaction module
Manufactured by Bio-Rad
Sourced in United States
The 96-Deep Well Reaction Module is a laboratory equipment designed for performing various reactions and experiments. It features a 96-well format with deep wells, allowing for the efficient handling and processing of multiple samples simultaneously. The module is a versatile tool suitable for a range of applications in research and development settings.
Automatically generated - may contain errors
Lab products found in correlation
C1000 touch thermal cycler, by Bio-Rad (15 mentions)
Chromium controller, by 10x Genomics (8 mentions)
Nextseq 500 sequencing system, by Illumina (5 mentions)
Agilent bioanalyzer, by Agilent Technologies (5 mentions)
Novaseq 6000, by Illumina (4 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (3 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (3 mentions)
High sensitivity dna kit, by Agilent Technologies (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (2 mentions)
Novaseq 6000 sequencing system, by Illumina (2 mentions)
Picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Chromium single cell controller, by 10x Genomics (1 mentions)
Chromium single cell 3 library gel bead kit, by 10x Genomics (1 mentions)
Qx200 droplet reader, by Bio-Rad (1 mentions)
Ddpcr kras screening multiplex kit, by Bio-Rad (1 mentions)
High sensitivity ngs analysis kit, by Agilent Technologies (1 mentions)
Gemcode single cell 3 gel bead and library v2 kit, by 10x Genomics (1 mentions)
Gemcode single cell instrument, by 10x Genomics (1 mentions)
18 protocols using 96 deep well reaction module
1
Chromium Single-Cell RNA-seq and V(D)J Sequencing
2
Bacterial DNA Isolation and 16S rRNA Sequencing
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Bacterial DNA from stool samples was isolated using a QIAamp stool DNA mini kit (QIAGEN, Hilden, Germany) by applying the method described in our previous report [39 (link)]. PCR of the V1-V3 region of the 16s rRNA gene sequences was carried out using a C1000 Touch thermal cycler with a 96-deep-well reaction module (Biorad, Hercules, CA, USA). The PCR products were purified using a LaboPass PCR purification kit (COSMO GENTECH, Seoul, Korea). The amplicons of each sample were pooled in equimolar amounts and then purified using AMPure XP beads (Agencourt Bioscience, Beverly, MA, USA) and finally quantified using a PicoGreen dsDNA assay kit (Invitrogen, Carlsbad, CA, USA). The mixed amplicons were amplified on sequencing beads by undertaking emulsion PCR. The sequencing reactions were conducted on a Roche/454 GS Junior system (454 Life Sciences, Branford, CT, USA) following the manufacturer’s instructions.
3
Single-cell RNA-Seq Library Construction
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Single cells were encapsulated into emulsion droplets using the Chromium Controller (10X Genomics). scRNA-Seq libraries were prepared using the Chromium Single Cell 3’ v2 Reagent Kit, following the manufacturer protocol. After sorting, sample volumes were reduced and cells were observed under a microscope and counted with a hemocytometer. Approximately 6,000 cells were then loaded into every channel. cDNA and library preparation was conducted on a C1000 Touch Thermal cycler with a 96-Deep Well Reaction Module (Bio-Rad). Amplified cDNA and final libraries were assessed using an Agilent BioAnalyzer and a High Sensitivity DNA Kit (Agilent Technologies). Individual libraries were diluted to 4 nM and combined for sequencing. The pools were sequenced with 75 cycle run kits (26 bp, read 1; 8 bp, index 1; and 55 bp, read 2) on the NextSeq 500 Sequencing System (Illumina), achieving an approximately 70%–80% saturation level.
4
Single-cell RNA-seq library preparation
5
Single-cell transcriptomics of in vitro germ cells
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FACS-sorted in vitro cells (hPGCLCs and d81 and d124 xrTestis samples) were collected in CELLOTION. hiPSCs and iMeLCs were collected in the StemFit Basic 04 and GK15 media, respectively, without FACS sorting. Cells were centrifuged at 300 × g for 5 min and then resuspended in 0.1% BSA in PBS.
Cells were loaded into chromium microfluidic chips with the Chromium Single Cell 3ʼ Reagent Kit (v3 chemistry) and then used to generate single-cell gel bead emulsions (GEMs) using the Chromium Controller (10× Genomics) according to the manufacturer’s protocol. GEM-RT was performed in a C1000 Touch Thermal Cycler with 96-Deep Well Reaction Module (Bio-Rad). All subsequent cDNA amplification and library construction steps were performed according to the manufacturer’s protocol. Libraries were sequenced using a 2 × 150 paired-end sequencing protocol on an Illumina HiSeq 4000 or NovaSeq 6000 instrument.
Cells were loaded into chromium microfluidic chips with the Chromium Single Cell 3ʼ Reagent Kit (v3 chemistry) and then used to generate single-cell gel bead emulsions (GEMs) using the Chromium Controller (10× Genomics) according to the manufacturer’s protocol. GEM-RT was performed in a C1000 Touch Thermal Cycler with 96-Deep Well Reaction Module (Bio-Rad). All subsequent cDNA amplification and library construction steps were performed according to the manufacturer’s protocol. Libraries were sequenced using a 2 × 150 paired-end sequencing protocol on an Illumina HiSeq 4000 or NovaSeq 6000 instrument.
6
Single-Cell RNA-Seq Using 10x Genomics
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Cell suspensions were loaded on a Chromium Single Cell Controller instrument (10× Genomics, Pleasanton, CA, USA) to generate single-cell gel beads in emulsions (GEMs). Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3′ Library & Gel Bead Kit (P/N 120237, 10× Genomics) according to the manufacturer’s instructions. Briefly, suspensions containing about 8000 cells per sample were mixed with RT-PCR reaction before being added to a chromium chip already loaded with barcoded beads and partitioning oil. The chromium chip was then placed in a Chromium Single Cell Controller instrument. GEM-RT-PCR was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad; CT022510) to produce barcode cDNA using the following program: 53 °C for 45 min; 85 °C for 5 min; maintain at 4 °C. Barcoded cDNA was isolated from the partitioning oil and then amplified by PCR. Sequencing libraries were generated from amplified cDNA using a 10× chromium kit, including reagents for fragmentation, sequencing adaptor ligation, and sample index PCR. Final libraries were sequenced on an Illumina Novaseq 6000.
7
Detecting KRAS Mutations via ddPCR
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Seven types of KRAS mutations (G12C, G12V, G12R, G12A, G12S, G12D, and G13D) were detected using the QX200 Droplet Digital PCR system (Bio-Rad). Commercial primers and probes were used ddPCR KRAS Screening Multiplex Kit (Bio-Rad). Briefly, up to 20,000 droplets were made from the master mix solution containing the probe, primers, template DNA, dNTPs, and DNA polymerase with the Cl000 Touch Thermal Cycler with a 96-Deep Well Reaction Module (Bio-Rad). PCR was performed using the following cycling conditions: polymerase activating step (10 min at 95°C), followed by 40 reaction cycles (30 sec at 95°C for denaturation and 1 min at 55°C for annealing and extension), and DNA polymerase deactivation step (10 min at 98°C). The PCR products were loaded onto a QX200™ Droplet Reader (Bio-Rad). The number of droplets containing mutant and wild-type alleles was counted using the QuantaSoft v.l.7.4 software (Bio-Rad). The variant allele frequency (VAF) was calculated as:
8
Single-cell sequencing for mouse transcriptome
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Single-cell sequencing for the tabula muris project was previously described [17 (link)]. In brief, single cells were captured in droplet emulsions using the GemCode Single-Cell Instrument (10x Genomics), and scRNA-seq libraries were constructed as per the 10x Genomics protocol using GemCode Single-Cell 3′ Gel Bead and Library V2 Kit. The samples were diluted in PBS with 2% FBS to a concentration of 1.000 cells per μL. Cells were loaded in each channel with a target output of 5.000 cells per sample. All reactions were performed in the BioRad C1000 Touch Thermal cycler with 96-Deep Well Reaction Module. Amplified cDNA and final libraries were evaluated on a Fragment Analyzer using a High Sensitivity NGS Analysis Kit (Advanced Analytical). Equal volumes of 16 libraries were pooled for sequencing on the NovaSeq 6000 Sequencing System (Illumina).
Sequencing of Fzt:DU and C57BL/6NRj samples were conducted by Genewiz (GENEWIZ Germany GmbH, Leipzig, Germany). Similar to the tabula muris project, single nuclei were captured in droplet emulsions on the 10xGenomics system and sequenced on the NovaSeq 6000 Sequencing System (Illumina, San Diego, CA, USA). In contrast to the tabula muris sequencing, cells were loaded with a target output of 10,000 cells per sample and the snRNA-seq libraries were constructed using Library V3 chemistry.
Sequencing of Fzt:DU and C57BL/6NRj samples were conducted by Genewiz (GENEWIZ Germany GmbH, Leipzig, Germany). Similar to the tabula muris project, single nuclei were captured in droplet emulsions on the 10xGenomics system and sequenced on the NovaSeq 6000 Sequencing System (Illumina, San Diego, CA, USA). In contrast to the tabula muris sequencing, cells were loaded with a target output of 10,000 cells per sample and the snRNA-seq libraries were constructed using Library V3 chemistry.
9
Single-Cell RNA Sequencing Protocol
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The following procedures were previously described. For single-cell sequencing, white blood cells were isolated with the Erythroclear Red Blood Cell Depletion Reagent Kit (STEMCELL Technologies, catalog no. 01738), according to the manufacturer’s protocol. Cells were washed with PBS with 0.02% bovine serum albumin. To obtain single-cell gel beads in emulsion (GEMs), we resuspended cells at a concentration of 1000 cells/μl and loaded the mix on the Chromium Comptroller Instrument (10x Genomics). Single-cell cDNAs and libraries were prepared with Chromium Single-Cell 3′ Library and Gel Bead Kit v3.1 (10x Genomics, catalog no. 1000121). Briefly, GEM-RT incubation was performed in a C1000 Touch thermal cycler with 96-deep well reaction module (Bio-Rad, catalog no. 1851197). Single-strand cDNAs were purified with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, catalog no. 37002D) and amplified with the C1000 cycler. Amplified cDNA products were cleaned with 0.6X DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, catalog no. 37002D). Quality and quantity of the cDNAs were assessed on 4200 TapeStation (Agilent Technologies) with High Sensitivity D5000 DNA ScreenTape (Agilent, catalog no. 5067-5592). Libraries were diluted to the same molarity and pooled for sequencing on NovaSeq6000 (Illumina) sequencers (23 (link)).
10
Quantifying HPV16 E6 and E7 Enrichment
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In order to determine the degree of target sequence enrichment in postcapture versus precapture libraries, we designed E6 and E7 primers/probe sets for dPCR. For HPV-16 E6: HPV16_E6_Forward, 5′-ACTGTGTCCTGAAGAAAAGCA; HPV16_E6_Reverse, 5′-GTCCACCGACCCCTTATATT; and a double quenched probe 5′-/56-FAM/ACATCTGGA/ZEN/CAAAAAGCAAAGATTCCA/3IABkFQ/. For HPV-16 E7: HPV16_E7_Forward, 5′-GAGGAGGATGAAATAGATGGTC; HPV16_E7_Reverse, 5′-CCGAAGCGTAGAGTCACA; and a double quenched probe 5′-/5HEX/TGGACAAGC/ZEN/AGAACCGGACA/3IABkFQ/. First, pre- and postcapture libraries were quantified using the dPCR Library Quantification Kit for Illumina Truseq (Bio-Rad, catalog no. 1863040). All dPCR reactions were carried out using a QX200 Droplet Digital PCR system (Bio-Rad). Thermocycling for 40 cycles was performed on a C1000 Touch Thermal Cycler with 96-Deep Well Reaction Module (Bio-Rad). Droplet analysis and copy-number quantification was performed using QuantaSoft software (Bio-Rad). We prepared dilutions of 2 ng/μL for precapture libraries and 0.02 pg/μL for postcapture libraries; 5 μL of each diluted library was used for E6 and E7 absolute quantification by dPCR. The degree of enrichment of the E6 and E7 sequences was determined by dividing the concentration in postcapture libraries by the concentration in the precaptured libraries.
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