Viably frozen CD138+-enriched or CD138+-depleted BM mononuclear cells were thawed at 37°C, resuspended in RPMI with 10% fetal bovine serum, and washed in cold phosphate-buffered saline, with cells being collected via centrifugation at 500g for 5 minutes. scRNA-seq was performed according to the Chromium Single Cell 3ʹ Reagent Kit version 2 user guide (10x Genomics). Generated gene expression libraries were pooled and paired-end sequenced (26 bp and 74 bp) on an Illumina NovaSeq 6000 S2. For scATAC-seq, cell pellets were carefully resuspended in an ice-cold NP-40 lysis buffer and spun down immediately. Nuclei were resuspended in the nuclei buffer provided by 10x Genomics, counted and subjected to Tn5 tagmentation. The subsequent steps were performed according to the manufacturer's instructions for the 10x Genomics Single Cell ATAC version 1.0. Generated libraries were pooled and paired-end sequenced (26 bp and 74 bp) on an Illumina NovaSeq 6000 S2.
Novaseq 6000 s2
Manufactured by Illumina
Sourced in United States, Germany
The NovaSeq 6000 S2 is a high-throughput sequencing system designed for large-scale genomic studies. It utilizes Illumina's proprietary sequencing-by-synthesis technology to generate high-quality sequencing data. The system is capable of processing multiple samples simultaneously, making it suitable for various genomic applications that require large-scale data generation.
Automatically generated - may contain errors
Lab products found in correlation
Ma900, by Sony (2 mentions)
Sh800, by Sony (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Dna hs kit, by Agilent Technologies (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Countess 2 cell counter, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Tapestation 4200, by Agilent Technologies (1 mentions)
Facsaria 2, by BD (1 mentions)
Isolate 2 genomic dna extraction kit, by Meridian Bioscience (1 mentions)
Truseq dna methylation kit, by Illumina (1 mentions)
Ex dna methylation lightning kit, by Zymo Research (1 mentions)
Rnase a, by Qiagen (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
I7 index, by Illumina (1 mentions)
Chromium single cell 3 library gel bead kit v2 or v3, by 10x Genomics (1 mentions)
Propidium iodide, by BioLegend (1 mentions)
25 protocols using novaseq 6000 s2
1
Single-Cell Multi-Omic Profiling of BM Cells
2
Retinal Organoid Single-Cell RNA-seq
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Distinct pools of H9 ES cell-derived retinal organoids (12–20 retinal organoids/pool) co-infected by LV-rtTA and LV-EGFP, LV-AEP, or LV-NEP were induced by Dox and dissociated between days 45 and 48 using trypsin and manual trituration. Dissociated cell suspensions were subjected to fluorescence activated cell sorting using FACSAriaII (BD Biosciences). Non-infected retinal organoid cells were used to set thresholds for selecting EGFP-positive cells. Sorted EGFP-positive cells were collected in HBSS without Ca2+ and Mg2+ (Thermo Fisher, 14170-112) containing 1% FBS and 0.4% BSA. The cells were washed with PBS containing 0.04% BSA, then counted with Countess II Cell Counter (Thermo Fisher).
Automated single-cell capture, barcoding, and cDNA library preparation were carried out using 10X Genomics Chromium Controller with Chromium Single Cell 3’ Library & Gel Bead Kit v2 reagents, with 12 cycles of cDNA amplification and 12 cycles of library amplification, following the manufacturer’s instructions. Qubit dsDNA Assay kit (Life Technologies) and TapeStation 4200 (Agilent) were used to assess the quality and concentration of the libraries. Illumina NovaSeq6000 S2 paired-end 2 × 50 bp mode was used to sequence the libraries.
Automated single-cell capture, barcoding, and cDNA library preparation were carried out using 10X Genomics Chromium Controller with Chromium Single Cell 3’ Library & Gel Bead Kit v2 reagents, with 12 cycles of cDNA amplification and 12 cycles of library amplification, following the manufacturer’s instructions. Qubit dsDNA Assay kit (Life Technologies) and TapeStation 4200 (Agilent) were used to assess the quality and concentration of the libraries. Illumina NovaSeq6000 S2 paired-end 2 × 50 bp mode was used to sequence the libraries.
3
Genome-Wide DNA Methylation Profiling of Koala Tissues
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Genomic DNA was extracted using a Bioline Isolate II Genomic DNA Extraction Kit (cat. no. BIO-52067) following the recommended protocol with an additional DNAse free RNaseA (100 mg ml−1) (Qiagen cat. no. 19101) treatment before column purification. Twenty milligram tissue samples from the brain, kidney, lung, skeletal muscle and pancreas from a female koala, ‘Pacific Chocolate’ (Australian Museum registration M.45022), and a male koala, ‘Ben’ (Australian Museum registration M.47723), were bisulfite converted using the EX DNA Methylation-Lightning Kit (Zymo cat. no. D5030). WGBS libraries were constructed using the TruSeq DNA methylation kit (Illumina cat. no. EGMK81213). The libraries were sequenced on a NovaSeq6000 S2 (Illumina) using the 2 × 100 bp PE option. Processing of the WGBS data followed previous studies [25 (link)]. Bisulfite conversion rates were estimated for each WGBS sample using methPipe's bsrate [45 (link)] (electronic supplementary material, table S1). Strand-specific methylation calls were combined, and all samples were filtered to remove CpGs covered by fewer than three reads (electronic supplementary material, table S1).
4
Single-Nucleus Transcriptome Analysis
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After cryostat dissection, samples were batched in groups of eight, chosen to include treated and control animals in every run. Single nucleus suspensions were prepared as described27 ,28 (link). Briefly: tissue samples were triturated, by pipetting, in an extraction buffer containing Kollidon VA64, Triton X-100, bovine serum albumin, and RNase inhibitor, then passed through a 26-gauge needle, washed and pelleted, then passed through a cell strainer. Nuclei positive for DAPI signal were isolated by fluorescence-activated cell sorting with a Sony SH800 or MA900 calibrated with a 70 μm chip, with a 405 nm excitation laser and light collected with a 425 – 475 nm filter. Sorted nuclei were counted using a Fuchs-Rosenthal C-Chip hemocytometer and a hand tally counter. A volume chosen to target 17,000 nuclei was submitted to the Broad Institute’s Genomics Platform, where 10X library construction (3’ V3.1 NextGEM with Dual Indexing) was performed according to manufacturer instructions29 . Libraries were sequenced on an Illumina Novaseq 6000 S2 for 100 cycles.
5
Single-Cell RNA-seq Library Preparation
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Sorted nuclei were processed using the 10× Chromium Next GEM Single Cell 3’ Kit v3.1 to generate the cDNA libraries. The quality of cDNA was assessed using the Agilent 2100 Bioanalyzer System. Sequencing was performed on Illumina NovaSeq 6000-S2.
6
Conditional Runx2 Deletion in Molars
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Gli1‐CreERT2;Runx2fl/fl and Runx2fl/fl littermate control mice received injection of tamoxifen at PN3.5 and were euthanized 4 days later. The apical halves of the mandibular first molars were dissected for RNA extraction. Each sample contained tissues from four mice. cDNA library preparation and sequencing were carried out by the Technology Center for Genomics & Bioinformatics at the University of California, Los Angeles (UCLA). A total of 200 million pair‐end reads were obtained on NovaSeq 6000 S2 (Illumina, San Diego, CA, USA) for four pairs of samples. The raw data was analyzed using Partek® Flow® software (Partek Incorporated, St. Louis, MO, USA). Briefly, raw reads were trimmed, aligned by STAR (2.6.1d;https://github.com/alexdobin/STAR/releases ) with the mm10 genome, and normalized using the fragments per kilobase of transcript per million mapped reads (FPKM) method. Differential analysis was performed using the gene set analysis (GSA) method. Value of p < 0.05 and fold change < −1.8 or >1.8 across groups were considered significant.
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Gli1‐CreERT2;Runx2fl/fl and Runx2fl/fl littermate control mice received injection of tamoxifen at PN3.5 and were euthanized 4 days later. The apical halves of the mandibular first molars were dissected for RNA extraction. Each sample contained tissues from four mice. cDNA library preparation and sequencing were carried out by the Technology Center for Genomics & Bioinformatics at the University of California, Los Angeles (UCLA). A total of 200 million pair‐end reads were obtained on NovaSeq 6000 S2 (Illumina, San Diego, CA, USA) for four pairs of samples. The raw data was analyzed using Partek® Flow® software (Partek Incorporated, St. Louis, MO, USA). Briefly, raw reads were trimmed, aligned by STAR (2.6.1d;
7
Single-cell RNA-seq Library Preparation
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Sorted nuclei were processed using the Chromium Next GEM Single Cell 3′ Kit v3.1 to generate the cDNA libraries. The quality of cDNA was assessed using the Agilent 2100 Bioanalyzer System. Sequencing was performed on Illumina NovaSeq 6000-S2.
8
Single Nucleus ATAC-Seq of Ocular Tissues
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Matching regions of the contralateral eyes of the sNucSeq donors, including sclera, choroid, and retina, were used for single-nucleus ATAC-seq using the Chromium Single Cell ATAC v1 kits (10X Genomics). This approach captures nuclei from both the macula and the periphery. Nuclei were isolated following a modified protocol based on Ziffra et al.112 (link) Frozen sections were lysed in a glass tissue homogenizer (Wheaton) in an ice-cold homogenization buffer (see above) without DAPI and RNase inhibitors. Released nuclei were washed in ice-cold wash buffer (10 mM Tris buffer pH 7.4, 100 mM NaCl, 3 mM MgCl2, 0.1% Tween 20, 1% BSA), filtered through FlowMi cell strainers (70 μm and 40 μm, Bel-Art), counted and resuspended in 1X Diluted Nuclei Buffer (10X Genomics), and loaded onto 10X Chromium Chip E using ∼15K nuclei per library. 2 libraries per donor were generated following the manufacturer’s protocol. Libraries were sequenced using Illumina HiSeq 2500 and NovaSeq 6000 S2 flow cell.
9
Single-cell RNA-seq Using 10X Chromium
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Single-cell RNAseq libraries were prepared using a Chromium Single Cell 3′ Library & Gel Bead Kit v2 or v3 (10X Genomics), according to the manufacturer’s protocol (user guide). Two chips were loaded with the accurate volumes calculated based on the 'Cell Suspension Volume Calculator Table.' The initial single-cell suspension being estimated at >600 cells/μl, we targeted to recover a maximum 10,000 cells. Once GEMs were obtained, reverse transcription and cDNA amplification steps were performed. Sequencing was done on Illumina NovaSeq 6000 S2 flow cell generating paired-end reads. Different sequencing cycles were performed for the different reads, R1 and R2. R1 contained 10× barcodes and UMIs, in addition to an Illumina i7 index, and R2 contained the transcript-specific sequences. All steps were performed at the Next Generation Sequencing platform at the University of Bern.
10
Single-cell analysis of wound healing in old mice
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