A single cell suspension of surgically resected human pituitary tumors was obtained by mechanical and enzymic digestion using a gentleMACS dissociator, and human tumor dissociation kit (Miltenyi Biotec Inc., Germany, Cat# 130-095-929). Library generation was performed on the 10x Genomics Chromium Controller following the manufacturer’s protocol for the v3 reagent kit (10x Genomics). In brief, cell suspensions were loaded onto a Chromium Single Cell A Chip, aiming for 10,000 cells per channel for generation of single-cell gel bead-in-emulsions (GEMs), following which reverse transcription was performed. The resulting post-GEM reverse transcription product was then cleaned using DynaBeads MyOne silane beads (Thermo Fisher Scientific, Waltham, MA). The cDNA was amplified, cleaned and quantified, then enzymatically fragmented and size selected prior to library construction. Libraries were quantified by KAPA quantitative PCR for Illumina adapters (Roche, Pleasanton, CA) and size was determined by Agilent TapeStation D1000 tapes. Libraries were sequenced on a NextSeq 500 sequencer (Illumina, San Diego, CA).
Genomics chromium controller
Manufactured by 10x Genomics
Sourced in United States
The 10x Genomics Chromium Controller is a laboratory instrument designed for automated sample preparation. It is used to generate single-cell libraries for various genomic applications, including single-cell RNA sequencing and single-cell immune profiling.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500 sequencer, by Illumina (3 mentions)
Novaseq, by Illumina (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
Chromium i7 multiplex kit, by 10x Genomics (3 mentions)
Novaseq 6000, by Illumina (2 mentions)
Chromium single cell a chip kit, by 10x Genomics (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Novaseq 6000 platform, by Illumina (2 mentions)
Nextseq 550, by Illumina (2 mentions)
Cryostore cs10, by STEMCELL (2 mentions)
Genomics chromium single cell atac library gel bead kit, by 10x Genomics (2 mentions)
Genomics platform, by 10x Genomics (2 mentions)
D1000 tapestation tape, by Agilent Technologies (1 mentions)
Dynabeads myone silane beads, by Thermo Fisher Scientific (1 mentions)
Human tumor dissociation kit, by Miltenyi Biotec (1 mentions)
S1000 touch thermal cycler, by Bio-Rad (1 mentions)
Chromium single cell b chip kit, by Genomics Plc (1 mentions)
Chromium single cell controller, by Genomics Plc (1 mentions)
Chromium microfluidic chip e, by 10x Genomics (1 mentions)
Novaseq 6000 sequencer, by Illumina (1 mentions)
24 protocols using genomics chromium controller
1
Single-Cell RNA-Seq Analysis of Human Pituitary Tumors
2
Single-cell nuclei sequencing
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Each 10,000 nuclei were used for the snRNA or snATAC library construction.
For snRNA-seq, the single cell 3′ GEM, Library & Gel Bead Kit V3.1 (10× Genomics, 1000075) and Chromium Single Cell B Chip Kit (10× Genomics, 1000074) were used. To generate single-nuclei gel beads in emulsion, the nuclei suspension was loaded onto the Chromium single cell controller (10× Genomics). Then suspended the single nuclei in PBS (containing 0.04% BSA). Captured cells were lysed to release their RNA and barcoded through reverse transcription in individual GEMs. The reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio-Rad) at 53 °C for 45 min, followed by 85 °C for 5 min. The cDNA was kept at 4 °C and then amplified for sequencing.
For snATAC-seq, incubating the nuclei with Tn5 transposase. Then the nuclei suspension was loaded into the Chromium microfluidic chip E with 10x Genomics reagents and barcoded with a 10x Genomics Chromium Controller (10x Genomics, Pleasanton, CA). DNA fragments were subsequently amplified, and the sequencing libraries were constructed with reagents from a Chromium Single Cell ATAC reagent kit (10x Genomics; PN-1000110, PN-1000156, PN-1000084) according to the manufacturer’s instructions. After preliminary quantification and quality inspection, libraries were then pooled and loaded on an Illumina NovaSeq with 2 × 50 paired-end kits.
For snRNA-seq, the single cell 3′ GEM, Library & Gel Bead Kit V3.1 (10× Genomics, 1000075) and Chromium Single Cell B Chip Kit (10× Genomics, 1000074) were used. To generate single-nuclei gel beads in emulsion, the nuclei suspension was loaded onto the Chromium single cell controller (10× Genomics). Then suspended the single nuclei in PBS (containing 0.04% BSA). Captured cells were lysed to release their RNA and barcoded through reverse transcription in individual GEMs. The reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio-Rad) at 53 °C for 45 min, followed by 85 °C for 5 min. The cDNA was kept at 4 °C and then amplified for sequencing.
For snATAC-seq, incubating the nuclei with Tn5 transposase. Then the nuclei suspension was loaded into the Chromium microfluidic chip E with 10x Genomics reagents and barcoded with a 10x Genomics Chromium Controller (10x Genomics, Pleasanton, CA). DNA fragments were subsequently amplified, and the sequencing libraries were constructed with reagents from a Chromium Single Cell ATAC reagent kit (10x Genomics; PN-1000110, PN-1000156, PN-1000084) according to the manufacturer’s instructions. After preliminary quantification and quality inspection, libraries were then pooled and loaded on an Illumina NovaSeq with 2 × 50 paired-end kits.
3
Single-Cell RNA-Sequencing with 10X Genomics
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ScRNA-seq libraries of 10X data were generated using the 10X Genomics Chromium Controller (10X Genomics) and Chromium Single Cell 3′ Reagent Kits v3.1 according to the manufacturer’s instructions. Reverse transcription, cDNA amplification, and sample indexing were performed using an Eppendorf Mastercycler. The final libraries were quantified using a KAPA library quantification kit (KK4824), and the fragment size distribution of the libraries was determined using a LabChip GX DNA high sensitivity kit (Perkin Elmer). Pooled libraries were then sequenced using a NovaSeq 6000 Illumina Sequencer with a NovaSeq 6000 S1 Reagent Kit (100 Cycles, 20012865).
4
Single-Cell Transcriptomics and Proteomics of PBMCs
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PBMCs were thawed and DNA-barcoded antibodies for CITE-seq were attached. Information on the antibodies used for CITE-seq is shown in Supplementary Table 9 . Single-cell suspensions were processed through the 10x Genomics Chromium Controller (10x Genomics). The libraries were constructed following the protocol outlined in the Chromium Single Cell 5’ Reagent Kits v2 (Dual Index, Cat. No. PN-1000263) User Guide (10x Genomics). Briefly, up to 10,000 labeled live cells per sample were separately loaded into the 10x Genomics platform without sample mixing to create a barcoded cDNA library for individual cells. Data quality control was performed using the Bioanalyzer (Agilent). Individual libraries were pooled for sequencing on the HiSeq 2500 or Novaseq 6000 platform (Illumina) to achieve at least 20,000 paired-end reads per cell for gene expression and 60,000 paired-end reads per cell for surface proteins. Sequence information is summarized in Supplementary Table 10 .
5
Single-Cell RNA-Seq of Mesp1 KO Cells
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Libraries for scRNA-seq were prepared according to manufacturer's instructions using the 10x Genomics Chromium controller, Chromium Single Cell 5′ Library and Gel Bead Kit v1 (10x Genomics, 1000006) and Chromium Single Cell A Chip Kit (10x Genomics, 1000151). A targeted maximum recovery of 10,000 cells per sample were loaded onto the 10x Genomics Chromium instrument, and each sample was indexed with a unique sample identifier (10x Genomics Chromium i7 Multiplex Kit, 120262). Final libraries were pooled and sequenced shallowly according to 10x protocol parameters on a NextSeq500 (Illumina), and then re-pooled for deeper sequencing on HighSeq4000 (Illumina) and/or NovaSeq using an S4 lane (Illumina). Littermate, stage-matched comparisons of control and Mesp1 KO libraries were always sequenced together in the same library pool. All scRNA-seq libraries were sequenced to a mean read depth of at least 50,000 total aligned reads per cell.
6
Single-cell Profiling of Human Airway Epithelium
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HAE cultures maintained for 3 weeks post-airlift were processed for scRNA-Seq. Single cell suspensions of HAE cultures were prepared as described previously27 (link). To enable multiplexing and doublet detection, cells were labeled with barcoded antibodies described previously28 (link). Briefly, approximately 200,000 cells per sample were resuspended in staining buffer (PBS, 2% BSA, and 0.01% Tween) and incubated for 10 minutes with Fc block (TruStain FcX (BioLegend) and FcR blocking reagent (Miltenyi). Cells were then incubated with oligonucleotide-conjugated hashing antibodies (generated in-house by the New York Genome Center as described29 ) for 30 min at 4°C. After labelling, cells were washed 3 times in staining buffer. After the final wash, cells were resuspended in PBS plus 0.04% BSA, filtered, and counted. Cells were pooled (6 samples per pool), super-loaded to the 10X Genomics Chromium Controller (~4,170 cells per sample, ~25,000 cells per lane) and processed with the Chromium Next GEM single-cell 5′ library and gel bead kit according to manufacturer’s protocols. Hashtag-oligonucleotide (HTO) additive oligonucleotide primer was spiked into the cDNA amplification PCR, and the HTO library was prepared as described previously29 .
7
Single-Cell RNA-Seq of Murine Heart
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Two replicates, with n=7 mice per treatment group, were used for heart sample processing. Approximately 16,000 cells were loaded onto a single channel of the 10X Genomics Chromium controller (10X Genomics, Pleasanton, CA), with a target recovery of ∼10,000 cells using the chromium v2 and v3 single-cell reagent kit. After the generation of single-cell gel bead-in-emulsions, cDNA was synthesized using a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) and amplified for 11 cycles as per the manufacturer’s protocol. Quality control (QC) and quantification were performed using the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) per the manufacturer’s guidelines. Amplified cDNA (50 ng) was used to construct 3’ expression libraries, and the libraries were pooled and run on an Illumina HiSeq 4000. Each lane consisted of 150 base-pair, paired-end reads. The Illumina basecall files were converted to FASTQ format, and these files were aligned to the murine genome (mm10) using the CellRanger v3.0.2 pipeline as described by the manufacturer. Across aligned cells, the mean number of reads per cell was 39,923, with an average of 95.3% of reads mapped to the mm10 genome.
8
Single-Cell RNA Sequencing of Thawed Cells
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We quickly thawed cells following retrieval from liquid nitrogen storage, washed cells twice in Dulbecco’s Modified Eagle Medium (Gibco), and treated them with 200 KU/ml DNaseI (Sigma). We counted live and dead cells using a dye exclusion assay with a Tecan automated counter. We adjusted the total cell concentration to 700–1200 cells/μL to be used for the 10x Genomics single-cell gene expression pipeline. We performed single-cell RNA sequencing on the microfluidic-based 10x Genomics platform according to manufacturer’s instructions. We used the Chromium Single Cell 3′v3.1 Reagent Kit. Briefly, we partitioned cells into individual droplets that contain a barcoded gel bead using the 10x Genomics Chromium Controller. We then lysed the cells and reverse transcribed RNA to cDNA. We broke the gel bead emulsion, amplified and fragmented cDNA, and added Illumina adapters. We sequenced samples on the Illumina NovaSeq 6000 platform at the McDonnell Genome Institute at Washington University School of Medicine in St. Louis.
9
Single-cell ATAC-seq analysis of skin ILCs
10
Isolation and Sequencing of Tumor-Infiltrating Leukocytes
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