The need for single cell sequencing is based on several factors.

The Chromium system would provide users with the latest and most advanced high-throughput sequencing technology available for single cell genomics. The sequencer will be placed in the Dartmouth Genomics & Molecular Biology Shared Resource (GMBSR), a core facility (1500 sq ft) in the Norris Cotton Cancer Center (NCCC) at the Geisel Medical School at Dartmouth with the expertise, knowledge, and experience to maximize the scientific potential of the new technology. (Pleasanton, CA) to carry out single-cell sequencing. Laboratory for lung development and regenrationĬomprehensive single cell RNA-seq datasets of mice trachea epithelial progenitors in 6 time points from E12.5 to E18.In order to meet the investigative needs of the Dartmouth research community in genomics, a request of $175,000 is submitted as part of a proposal to purchase a Chromium instrument from 10X Genomics, Inc. Supplementary_files_format_and_content: Filtered gene-barcode matrix of CellRanger output including sparse matrix (MEX), cellular barcodes (TSV), and gene list (TSV). To maintain a standard procedure for clustering, a value of 0.8 for the resolution was used

Top 15 principal components (PCs) were selected by using a permutation-based test implemented in Seurat and passed to t-Distributed Stochastic Neighbor Embedding (tSNE) for clustering visualization. For clustering, principal-component analysis was performed for dimension reduction. Seurat v2.3.4: the cells meeting any of the following criteria were omitted from further analyses for the quality control 5,000 UMIs, > 7.5% of reads mapping to mitochondria genes, or EpCAM negative cells. Signle cell RNA-seq reads were aligned to the mm10 genome reference using Cellranger v2.2.0 with default parameters Single cell RNA-seq using Chromium system (PolyA+ RNA)īasecalls performed using Illumina RTA 1.18.64 and the fastq files were generated by Cellranger v2.2.0 and bcl2fastq 2.19.0.316 Libraries were sequenced on a HiSeq1500 (Illumina) to obtain a sequencing depth of around 50,000 reads per cells.

Indexed sequencing libraries were constructed using Chromium Single Cell 3′ Library v2 (10X Genomics, PN-120233) for enzymatic fragmentation, end-repair, A-tailing, adaptor ligation, ligation cleanup, sample index PCR, and PCR cleanup. After performing GEM-reverse transcriptions (GEM-RTs), GEMs were harvested and the cDNAs were amplified and cleaned up with SPRIselect Reagent Kit (Beckman Coulter, B23318). Briefly, single-cell gel bead-in-emulsions (GEMs) were generated from loaded cell suspensions by a Chromium Controller instrument (10X Genomics). Signle-cell suspensions were loaded onto Chromium Single Cell A Chips (10X Genomics, PN-1000009) for the Chromium Single Cell 3′ Library v2 (10X Genomics, PN-120233) according to the manufacturer’s recommendations (10X Genomics). Single-cell suspensions were made through the incubation with Trypsin-EDTA (0.25%) (Thermo Fisher Scientific, 25200056) at 37℃ for 15 min, then loaded onto Chromium Single Cell A Chips (10X Genomics, PN-1000009) for the Chromium Single Cell 3′ Library v2 (10X Genomics, PN-120233) according to the manufacturer’s recommendations (10X Genomics) To prepare single-cell suspensions of epithelial cells in 7 time points (E12.5, E13.5, E14.5, E16.5, E18.5, and 2months), the epithelial sheet was peeled off from mesenchymal tissue in developing trachea with a tungsten needle after the incubation with 175U/ml collagenase typeⅠ (Worthington Biochemical Corporation, CLS1) at 37℃ for 6 - 120 min. GEO help: Mouse over screen elements for information.

10x chromium system