Lungs from tumor-bearing mice treated with either FLT3L and αCD40, or rat IgG2a control isotype were harvested 9 days after tumor injection. Single cell suspension was obtained using gentle MACS dissociator (Milteny), filtered using 70 µm cell strainer. Samples were incubated with α-CD45-APCFire and Live/Dead Fixable Aqua Staining (Life technologies) to asses cell viability. CD45+ cells were isolated using ARIA Cell Sorter (BD). Single cells were prepared using Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) (10X Genomics), following the manufacturer’s instructions. In brief, 10,000 cells/channel were partitioned on Chromium Next GEM Chip G using the Controller platform (10X Genomics). After encapsulation and simultaneous cell barcoding, library preparation was performed. Following quality controls, libraries were sequenced according to manufacturer’s instruction on Novaseq6000 platform (Illumina). Target coverage was 50,000 cluster/cell.
Chromium next gem single cell 3 reagent kits v3.1 dual index
Manufactured by 10x Genomics
The Chromium Next GEM Single Cell 3' Reagent Kits v3.1 Dual Index are laboratory equipment designed for single-cell RNA sequencing applications. The kits provide the necessary reagents and consumables to generate and process single-cell libraries from a variety of sample types. The kits are compatible with the Chromium Controller instrument from 10x Genomics.
Automatically generated - may contain errors
Lab products found in correlation
Tapestation 4150, by Agilent Technologies (3 mentions)
Cell free dna screentape assay, by Agilent Technologies (3 mentions)
Hiseq x, by Illumina (3 mentions)
Novaseq 6000, by Illumina (2 mentions)
Live dead fixable aqua staining, by Thermo Fisher Scientific (1 mentions)
Chromium next gem chip g, by 10x Genomics (1 mentions)
Aria cell sorter, by BD (1 mentions)
Novaseq 6000 platform, by Illumina (1 mentions)
Mouse lung dissociation kit, by Miltenyi Biotec (1 mentions)
Mouse spleen dissociation kit, by Miltenyi Biotec (1 mentions)
Qubit 1x dsdna high sensitivity assay, by Thermo Fisher Scientific (1 mentions)
Chromium controller, by 10x Genomics (1 mentions)
Evos m7000 microscope, by Thermo Fisher Scientific (1 mentions)
Readyprobes cell viability imaging kit blue green, by Thermo Fisher Scientific (1 mentions)
Fragment analyzer system, by Agilent Technologies (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
8 protocols using chromium next gem single cell 3 reagent kits v3.1 dual index
1
Transcriptomic Profiling of Tumor-Bearing Murine Lungs
2
Mouse Lung and Spleen Dissociation
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We used Mouse lung dissociation kit (130-095-927) and Mouse spleen dissociation kit (130-095-926) that were purchased from Miltenyi Biotec and are commercially available. Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) (CG000315) used for library preparation was purchased from 10X Genomics and is available commercially.
3
Single-cell RNA-seq of mouse mammary gland
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Mammary gland from 8-week-old mice (one p53R172H/R172H knock-in mouse and one p53+/+ mouse) were isolated and digested as previously described98 (link). scRNAseq libraries were prepared by using Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 Dual Index (10X Genomics) following manufacturer’s instructions. Estimated 16,500 cells were loaded to each channel with the average recovery rate of 10,000 cells. Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate size fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. The final indexed libraries were quantified by using the Cell-free DNA Screen Tape Assay (Agilent) on a TapeStation 4150 (Agilent) and were sequenced on HiSeqX (Illumina).
4
Single-cell RNA-seq of mouse mammary gland
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Mammary gland from 8-week-old mice (one p53R172H/R172H knock-in mouse and one p53+/+ mouse) were isolated and digested as previously described98 (link). scRNAseq libraries were prepared by using Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 Dual Index (10X Genomics) following manufacturer’s instructions. Estimated 16,500 cells were loaded to each channel with the average recovery rate of 10,000 cells. Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate size fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. The final indexed libraries were quantified by using the Cell-free DNA Screen Tape Assay (Agilent) on a TapeStation 4150 (Agilent) and were sequenced on HiSeqX (Illumina).
5
Single-cell RNA-seq of let-7a-5p+ imMKCLs
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imMKCLs (clone 7) with distinct let-7a-5p activity were sorted separately and resuspended at 1000 cells/μL in PBS with 0.4% BSA (Fig. 3 ). The cells were loaded onto a Chromium Next Gel Beads-in-EMulsion (GEMs) Chip G Single Cell Kit (10 x Genomics, USA). GEM generation and barcoding, reverse transcription, and cDNA generation and library construction were performed following the manufacturer’s protocol (Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 Dual Index, 10 x Genomics). Dual-indexed, single-cell libraries were pooled and sequenced in paired-end reads on a Novaseq6000 (Illumina).
6
Single-Cell RNA-seq of Bone Cells
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Single cells isolated from digested bones were stained with ReadyProbes cell viability imaging kit, blue/green (Thermo Fisher Scientific, catalog # R37609) and manually counted using a hemocytometer under an EVOS M7000 microscope (Thermo Fisher Scientific). Immediately following cell counting, samples were processed using Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index) as described in the manufacturer’s instructions (10X Genomics). In brief, aiming for 8000 cells per library, single-cell suspensions with more than 70% live cells were loaded onto Chromium Controller (10X Genomics) to generate gel beads-in-emulsions. Then, copartitioned cells were lysed, primers were released from gel beads, and barcoded full-length cDNA was produced and amplified. 3′ gene expression libraries were generated from cDNA by fragmentation, end repair, A-tailing, adaptor ligation, and index PCR amplification. The concentration and size distribution of final libraries were assessed by Qubit 1X dsDNA high sensitivity assay (Thermo Fisher Scientific, catalog # Q33231) and the fragment analyzer system (Agilent). Libraries were sequenced either on a NextSeq500 or NovaSeq 6000 (Illumina) with paired-end mode (read1: 28 cycles, read 2: 90 cycles, i7: 10 cycles, i5: 10 cycles) to generate a minimum of 20,000 read pairs per cell.
7
Single-Cell RNA Sequencing of Cell Subsets
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Reporter or cell surface marker-positive cells were sorted by BD FACSAria III Cell Sorter and loaded according to the manufacturer’s protocol for the Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index) (10x Genomics) to attain between 2,000 and 6,000 cells per reaction. Library preparation was carried out according to the manufacturer’s protocol. Libraries were sequenced, aiming at a minimum coverage of 40,000 raw reads per cell, on the Novaseq 6000 systems using the sequencing format: read 1, 28 cycles; i7 index, 10 cycles; i5 index, 10 cycles; read 2, 90 cycles.
8
Single-cell RNA-seq of mouse mammary gland
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Mammary gland from 8-week-old mice (one p53R172H/R172H knock-in mouse and one p53+/+ mouse) were isolated and digested as previously described98 (link). scRNAseq libraries were prepared by using Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 Dual Index (10X Genomics) following manufacturer’s instructions. Estimated 16,500 cells were loaded to each channel with the average recovery rate of 10,000 cells. Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate size fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. The final indexed libraries were quantified by using the Cell-free DNA Screen Tape Assay (Agilent) on a TapeStation 4150 (Agilent) and were sequenced on HiSeqX (Illumina).
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