All single cells were captured using the 10X Chromium controller (10X Genomics) according to the manufacturer’s specifications and manual (Manual CG000183, Rev A). Cells from each scRNA-seq sample (one iPSC, seven fresh iPSC-PPCs, RNA_Pool_1, and RNA_Pool_2) were loaded each onto an individual lane of a Chromium Single Cell Chip B. Libraries were generated using Chromium Single Cell 3’ Library Gel Bead Kit v3 (10X Genomics) following manufacturer’s manual with small modifications. Specifically, the purified cDNA was eluted in 24 μl of Buffer EB, half of which was used for the subsequent step of the library construction. cDNA was amplified for 10 cycles and libraries were amplified for 8 cycles. All libraries were sequenced on a HiSeq 4000 using custom programs (fresh: 28-8-175 Pair End and cryopreserved: 28-8-98 Pair End). Specifically, eight libraries generated from fresh samples (one iPSC and seven iPSC-PPC samples) were pooled together and loaded evenly onto eight lanes and sequenced to an average depth of 163 million reads. The two libraries from seven cryopreserved lines (RNA_Pool_1 and RNA_Pool_2) were each sequenced on an individual lane to an average depth of 265 million reads. In total, we captured 99,819 cells. We observed highly correlated cell type proportions between fresh and cryopreserved iPSC-PPC samples (Supplementary Fig. 8 ).
Chromium single cell b chip
Manufactured by 10x Genomics
Sourced in United States
The Chromium Single Cell B Chip is a microfluidic device designed for single-cell genomic analysis. It is a core component of the Chromium platform, enabling the encapsulation of individual cells into nanoliter-scale gel beads for subsequent library preparation and sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Chromium i7 multiplex kit, by 10x Genomics (5 mentions)
Chromium single cell 3 library gel bead kit v3, by 10x Genomics (4 mentions)
Chromium single cell controller, by 10x Genomics (4 mentions)
Novaseq 6000 system, by Illumina (3 mentions)
Nextseq 500, by Illumina (3 mentions)
Chromium controller, by 10x Genomics (2 mentions)
Chromium single cell 3 reagent kits v3, by 10x Genomics (2 mentions)
Fragment analyzer, by Agilent Technologies (2 mentions)
Ngs fragment kit, by Agilent Technologies (2 mentions)
Chromium single cell 3 gel beads, by 10x Genomics (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Novaseq sequencing platform, by Illumina (1 mentions)
Chromium single cell 3 reagent kit v3, by 10x Genomics (1 mentions)
Dnase, by STEMCELL (1 mentions)
Versene, by Lonza (1 mentions)
Tc10 cell counter, by Bio-Rad (1 mentions)
Cd45 til microbeads, by Miltenyi Biotec (1 mentions)
Cell ranger, by 10x Genomics (1 mentions)
Partitioning oil, by 10x Genomics (1 mentions)
16 protocols using chromium single cell b chip
1
Single-cell RNA-seq of iPSCs and iPSC-PPCs
2
Chromium Single Cell 3' Sequencing
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Construction of scRNA-seq libraries was strictly performed according to the instructions utilizing Chromium Single Cell 3' Reagent Kits v3 (PN-1000075, PN-1000073, PN-120262). Cell suspensions containing than 85% viable cells, less than 10% doublets and no large cell aggregates were identified as qualified samples. According to cell counting results, cells were resuspended at 1,000 cells/mL to prepare the reaction mix. After loading the Chromium Single Cell Chip B (10× Genomics, 2000060), the Chromium Controller was used to generate the Gel Bead-in-Emulsion (GEMs). Immediately following that, the reverse transcription (RT) was performed to produce the barcoded, full-length cDNA from poly-adenylated mRNA released from the captured cells. Then, the first-strand cDNA was purified and amplified to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. After sequential adding of P5, P7, a sample index and TruSeq Read 2, the libraries were constructed. After quality control by Agilent 2100 Bioanalyzer, the libraries were sequenced on Illumina Hiseq X ten PE150 platform in 150 bp pair-ended manner (Novogene Technology Co., Ltd., Tianjin, China).
3
Single-Cell RNA-Seq with Chromium Platform
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The nuclear suspensions were loaded onto Chromium Single-Cell Chip B (10X Genomics, USA) to generate single-cell Gel Beads-in-Emulsion (GEMs) using the Chromium Single-Cell Controller to capture 5 000–8 000 single cells per reaction. Reverse transcription (first-strand cDNA generation), full-length cDNA amplification, and 3’ library construction were conducted using a Chromium Single-Cell 3’ Reagent Kit v3 (10X Genomics, USA) following the manufacturer’s user guide. The libraries were sequenced on the Illumina NovaSeq sequencing platform with a paired-end read length of 150 bp.
4
Generating Single-Cell Sequencing Data from EPS-Blastoids
5
Single-cell profiling of MC38 tumor
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For MC38 tumor inoculations, 0.25 x 106 MC38 tumor cells were subcutaneously injected into C57BL/6 mice on day 0. One or ten days after implantation mice received 100 mpk dosing of PF-06821497 or vehicle control, subcutaneous, daily. CD45+ cells were isolated using CD45 TIL microbeads (Miltenyi, 130-110-618) and libraries were generated using the Single Cell 3’ GEM, Library and Gel Bead Kit v3 (10X Genomics, PN-1000075), Chromium Single Cell B Chip (PN-100073), and Chromium i7 Multiplex Kit (PN-120262). Sequencing was performed by Novogene and downstream analysis performed using Seurat. Initial processing of scRNA-Seq data was performed using Cell Ranger (10X Genomics) and then further analyzed using Seurat. A cut-off of greater than 200 genes and less than 5000 genes per cell was used to filter samples. Cells with UMI greater than 10% that were attributable to mitochondrial genes were excluded. Genes were only analyzed if they were present in a minimum of three cells. Normalizing and scaling was performed using the default settings in Seurat. A total of 29559 (day 7 vehicle), 71848 (day 7 EZH2i), 23404 (day 16 vehicle), and 13757 (day 16 EZH2i) were pooled and used for analysis after initial QC. Subsequent reclustering of lymphoid cells was performed.
6
Single-Cell RNA-Seq Library Preparation
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A single cell/nuclei suspension containing 10,000 cells/nuclei was loaded on a Chromium Single Cell B Chip (10x Genomics, catalog no. 1000154) as follows: 75 μL of master mix + nuclei suspension was loaded into the row labeled 1, 40 μL of Chromium Single Cell 3 Gel Beads (10x Genomics, catalog no. PN-1000093) into the row labeled 2 and 280 μL of Partitioning Oil (10x Genomics, catalog no. 2000190) into the row labeled 3. This was followed by GEM generation and barcoding, post GEM-RT cleanup and cDNA amplification. Then 100 ng purified cDNA derived from 12 cycles of cDNA amplification was used for 3 Gene Expression Library Construction by using Chromium Single Cell 3 GEM, Library & Gel Bead Kit v3 (10x Genomics, catalog no. 1000092) according to the manufacturer’s manual (10x Genomics, catalog no. CG000183 Rev C). The barcoded short-read libraries were measured using a Qubit 2.0 with a Qubit dsDNA HS assay kit (Invitrogen, catalog no. Q32854) and the quality of the libraries was assessed on a Fragment analyzer (Agilent) using a high-sensitivity NGS Fragment Kit (1–6000bp) (Agilent, catalog no. DNF-474-0500). Sequencing libraries were loaded on an Illumina NovaSeq6000 with PE 2 × 50 paired-end kits by using the following read length: 28 cycles Read1, 8 cycles i7 index and 91 cycles Read2.
7
Single-cell RNA-seq of Human Samples
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Single-cell RNA-sequencing was performed using the chromium single-cell 3ʹ RNA-sequencing system (10× Genomics, Pleasanton, CA) with the Reagent Kit v3 according to the manufacturer’s instructions. Briefly, the cells were loaded into chromium single cell B chip (10× Genomics, Pleasanton, CA) and gel beads in emulsion (GEM) generation were performed aiming at 10 000 cell capture per sample. Subsequent cDNA purification, amplification, and library construction were performed as instructed. Sample libraries were sequenced on an Illumina NovaSeq 6000 system using following read lengths: 28 bp (Read 1), 8 bp (i7 Index), 0 bp (i5 Index), and 89 bp (Read 2) aiming at 50 000 reads per cell sequencing depth.
The Cell Ranger v3.1 mkfastq and count pipelines (10× Genomics, Pleasanton, CA) were used to demultiplex and convert chromium single-cell 3ʹ RNA-sequencing barcodes and read data to FASTQ files and to generate aligned reads and gene-cell matrices. Reads were aligned to the human genome GRCh38.
The Cell Ranger v3.1 mkfastq and count pipelines (10× Genomics, Pleasanton, CA) were used to demultiplex and convert chromium single-cell 3ʹ RNA-sequencing barcodes and read data to FASTQ files and to generate aligned reads and gene-cell matrices. Reads were aligned to the human genome GRCh38.
8
Striatal Neuron Nuclei Isolation and FACS
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On DIV 11, striatal primary cells cultured on 12-well plates (250 K per well) were treated with Neurobasal vehicle, 50 μM DA, 1 μM SKF-38393, or 25 mM KCl and placed back into the incubator for 1 hour. Cells were then washed with 1 ml of PBS per well, followed by a 5-min incubation period in 600 μl of lysis buffer. Cells were then gently pipette mixed five times with a P1000 before being transferred to a 1.5-ml tube and centrifuged at 500 rcf for 5 min at 4°C. Supernatant was then removed, and the pellet was resuspended in 100 μl of nuclei wash buffer. Tubes for each treatment group were then combined, gently pipette-mixed five times, and passed through a 40 μM filter before FACS to further purify nuclei for sequencing. After FACS, 2400 nuclei per treatment condition were loaded into individual wells of the Chromium Single Cell B Chip (10x Genomics, catalog no. 1000074) using four of the eight available wells. Chromium single-nuclei capture and barcoding were completed on the Chromium Single Cell Controller according to manufacturer’s instructions.
9
High-throughput Single-cell RNA Sequencing
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The final concentration of single-cell suspension was adjusted to 1,000 cells/μl. In order to recover 8,000-10,000 cells, a volume of 15 µl was loaded into one channel of the Chromium™ Single Cell B Chip (#1000073, 10x Genomics). Single-cell bar-coding, cDNA synthesis and library preparation were performed by using the Chromium Single Cell 3′ Library & Gel Bead Kit v3 (#1000075, 10x Genomics). DNA libraries were then sequenced on an Illumina novaseq 6000 system configured with the paired-end 150-bp protocol for a sequencing depth of approximately 400 million reads per library.
10
Single-Cell RNA-Seq Library Preparation
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The final concentration of single‐cell suspension was adjusted to 1000 cells/μl, and a volume of 15 µl was loaded into one channel of the Chromium™ Single Cell B Chip (#1000073, 10x Genomics), aiming at recovering 8000–10,000 cells. The Chromium Single Cell 3’ Library & Gel Bead Kit v3 (#1000075, 10x Genomics) was used for single‐cell bar‐coding, cDNA synthesis and library preparation, following the manufacturer's instructions provided as the Single Cell 3’ Reagent Kits User Guide Version 3. Library sequencing was performed on a NovaSeq 6000 system (Illumina) configured with the paired‐end 150 bp protocol at a sequencing depth of approximately 400 million reads.
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