Single cells were captured using the Chromium Single Cell 3’ kit (10X Genomics, PN-1000075), according to the manufacturer’s recommendations. We aimed for 10,000 cells per library whenever possible. Both v2- and v3-chemistry were used for data presented here. For simultaneous detection of transcriptome and CRISPR-induced genetic scar sequences for lineage tracing, we proceeded as previously described24 . In brief, the scar sequences were enriched by a two-round nested PCR approach, using 10μl of the 10X Genomics cDNA and target site specific primers (dTomato transgene). The scar library was then indexed using the indexing primers of the Chromium kit to make sure they can be sequenced and demultiplexed together with the transcriptome. Samples were sequenced on Illumina NextSeq 500 150 bp and Illumina HiSeq 2500 200 bp after successful quality control by Bioanalyzer (DNA HS kit, Agilent).
Bioanalyzer hs dna kit
Manufactured by Agilent Technologies
The Bioanalyzer HS DNA Kit is a lab equipment product from Agilent Technologies. It is used for the analysis and quantification of DNA samples, providing high-sensitivity detection and accurate sizing of DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Chromium single cell 3 kit, by 10x Genomics (2 mentions)
Atac seq kit, by Active Motif (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Miseq sequencer, by Illumina (1 mentions)
Nextera xt library prep kit, by Illumina (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Pippinht, by Sage Science (1 mentions)
Nextflex dna barcodes, by PerkinElmer (1 mentions)
Nextseq 500 instrument, by Illumina (1 mentions)
Ampure xp system, by Beckman Coulter (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
7 protocols using bioanalyzer hs dna kit
1
Single-Cell Transcriptome and Lineage Tracing
2
Single-cell RNA-seq of Injured Fish Hearts
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Single cells were captured using the Chromium Single Cell 3' kit (10X Genomics, PN-1000075), according to the manufacturer's recommendations. We aimed for 10,000 cells per library whenever possible. Both v2-and v3-chemistry were used for data presented here. Samples (3 untreated and 3 morphine-treated hearts of injured fish at 3, 7, 15dpi) were sequenced on Illumina NextSeq 500 2x 75 bp and Illumina HiSeq 2500 2x 100 bp after successful quality control by Bioanalyzer (DNA HS kit, Agilent).
3
ATAC-seq Library Preparation for Lens Epithelial Cells
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ATAC-seq library preparation was performed using the ATAC-Seq Kit (Active Motif cat.53150) per the manufacturer’s instructions. Briefly, four lens epithelial explants per sample (biological replicate) were collected in ice-cold PBS. Two biological replicates were conducted for each ATAC-seq condition. The samples were centrifuged at 8000× g for 5 min at 4 °C. 100 µL of ATAC lysis buffer was added to the samples after aspirating the PBS. The explant samples were pipetted vigorously until visibly broken. The total number and quality (round shape without blebbing) of isolated nuclei was accessed by trypan blue staining that ranged between 80,000–120,000 per sample. Following nuclei validation, the samples were centrifuged, tagmented and PCR amplified. The amplified DNA was purified and indexed (Dual indexing following manufacturer protocol) to create sequence-ready libraries. The libraries were quantified using a Qubit 4 Fluorometer with the dsDNA Quantitation, high sensitivity kit (ThermoFisher Scientific, cat. Q32851). Final libraries were quality validated using the Bioanalyzer HS DNA Kit (Agilent, cat. 5067-4626). Validated libraries passing in-house quality control were sent to Novogene (Sacramento, CA, USA) for sequencing with a HiSeq platform using 150 base paired-end reads to a depth of greater than 30 million reads.
4
Bacterial DNA Extraction and Sequencing
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Total DNA extraction was performed on 1 mL of overnight cultures prepared as described above using DNeasy Blood & Tissue kit (Qiagen, Milan, Italy), following manufacturer’s instructions for lysis of Gram+ bacterial cells. Plasmid DNA extraction was performed on 1 mL overnight cultures with Plasmid Mini Prep kit (Fisher Molecular Biology, Rome), following the manufacturer’s instructions. An amount of 1 ng DNA from each sample was used to prepare Next Generation Sequencing libraries with the Illumina Nextera XT library prep kit. Quality control of the libraries was performed with Agilent Bioanalyzer HS DNA kit and quantitation with Qubit DNA HS kit. Approximately 4 Million 2x150 bp PE reads were generated for each sample in Illumina MiSeq Sequencer.
5
Illumina Library Preparation and Sequencing
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Libraries were prepared using the KAPA HTP Library Prep Kit for Illumina (Roche, KK8234) and Bioo Scientific NEXTflex DNA barcodes (Perkin Elmer, NOVA-514104). The resulting libraries were purified using the Agencourt AMPure XP system (Beckman Coulter, A63882) then quantified using a Bioanalyzer HS DNA kit (Agilent Technologies, 5067–4626) and a Qubit fluorometer (Life Technologies) with Qubit dsDNA HS assay kit (Invitrogen, Q32851). Post amplification size selection (275–700 bp) was performed on all libraries using a PippinHT (Sage Science) using 1.5% cassette (HTC1510). Libraries were pooled, requantified, and sequenced as 150 bp paired reads on a mid-output flow cell using the Illumina NextSeq 500 instrument. Following sequencing, Illumina Real Time Analysis version RTA 2.4.11 and bcl2fastq2 v2.20 were run to demultiplex reads and generate FASTQ files.
6
Single-cell RNA-seq of Forskolin-treated Cells
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Forskolin daily and alternate treated cells were taken for library preparation using the 10x genomics Chromium Next GEM Single Cell 3 0 Reagent Kit v3.1. The single-cell RNA libraries were prepared for both samples using the 10x genomics Chromium Next GEM Single Cell 3 0 Reagent Kit v3.1. The library QC and quantification was done using Agilent bioanalyzer HS DNA kit. The libraries were pooled and sequenced on the NextSeq 2000 platform. Raw bcl files were converted to final count matrix using Cell Ranger v6.1.2 software following the tutorial provided on the 10x genomics website. All the downstream analysis was performed in R Studio using Seurat v4 package. Low-quality cells and genes expressing in less than 3 cells were filtered out. The data was log normalised and 3,000 highly variable genes and 22 principal components were used for dimensional reduction and clustering.
7
ATAC-seq RPE Nuclei Profiling
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ATAC-seq library preparation was performed using ATAC-Seq Kit (Active Motif cat. 53150) per manufacturer’s instructions. Briefly, RPE was isolated in cold PBS as described above for RNA collection. Two isolated sheets of RPE were collected per biological sample and placed on ice. RPE cells were lysed in cold ATAC-Seq Kit lysis buffer and nuclei were counted with a hemocytometer. Approximately 100,000 RPE nuclei from two embryos were aliquoted for Tn5 tagmentation per sample. Final libraries were quality validated using the Bioanalyzer HS DNA Kit (Agilent, cat. 5067-4626) and quantified using the Qubit 4 Fluorometer with the dsDNA Quantitation, high sensitivity kit (ThermoFisher Scientific, cat. Q32851). Validated libraries were sequenced at the Novogene sequencing core (Sacramento, CA, United States) on an Illumina HiSeq Series sequencing platform to a minimum depth of 61 million read pairs per sample using 150 base pair reads.
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