![]() ![]() When analyzing high molecular weight DNA samples the linked read sequencing data delineate linkage information over distances of up to 150 kb. The sequencing libraries are generated with the Chromium controller instrument and reagents from 10X Genomics, followed by sequencing on our Illumina HiSeq sequencers. We are offering 10X Genomics Chromium Genome “linked read” library preps and sequencing. Please contact to reserve reagents for your project. ![]() As soon as these run out, we will discontinue the assay effective immediately. Update July 2020: We have ~16 remaining 10X Chromium Genome reactions left in our core. We are sad to see this very popular assay go. 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.As of January 2020, 10X Genomics has announced that it is discontinuing production of the 10X Chromium Genome linked read reagents. 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. ![]()
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