Row names are Ensembl_IDs, with each sample included as columns. Supplementary_files_format_and_content: Read count quantitation file was generated by counting reads using Rsubread, normalisation with RUVseq package (k = 3, with model.matrix ~d$sex +d$group) followed by vsd normalisation using ‘varianceStabilzingTransformation’ within DESeq2 package. BAM files were imported to Seqmonk v1.45.0. They were mapped to the human GRCh38 genome assembly including ERCC spike-ins using HISAT2 2.1.0 (no soft clipping). You can also download Visio Viewer for iOS to view and interact with the. Reads were trimmed using Trim Galore v0.4.4 ( ) using default parameters to remove the standard Illumina adapter sequence. From Redmond, they want to preach by example and today the tool Visio has. Libraries were sequenced on the Illumina HiSeq 2500 platform, with 6 samples pooled per lane. Library quantitatation was performed using Agilent Bioanalyser, KAPA qPCR and Qubit High-sensitivity kits. Libraries were generated from amplified cDNA using the NexteraXTDNA library preparation kit from Illumina, with nexteraXT indexes. Quality of cDNA was confirmed using an Agilent Bioanalyser. Between 16 and 18 PCR cycles were used for each sample, depending on cell input. ![]() Cells from day 7 after vaccination (cTfhTet) were also sorted on the basis of expressing PD1 and CXCR5 markers.ĬDNA was prepared directly from cells (40-1000 max) lysed in 1x lysis buffer, and amplified acccording to the protocol for the SmartseqV4 Ultra low input RNA kit for sequencing from Takara Clontech. Live CD3+ CD4+ CD45RA- TET+ cells were sorted on a BD Aria Fusion sorter directly into 1x lysis buffer as per the SmartseqV4 Ultra low input RNA kit for sequencing from Takara Clontech. Before sorting, ~10-30 million PBMC cells were thawed in culture media and rested for 2 hours at 37 degrees C, after which influenza HLADR tetramers (TET)added along with a panel of cell surface antibodies. After downloading and installing Microsoft Visio 2016 Viewer, when double-clicking the drawing file (.vsd) in Windows Explorer, it opens Internet Explorer and Visio Viewer renders the drawing in the browser window. ![]() PBMCs were isolated at the Babraham Institute by density gradient centrifugation and cryopreserved in liquid nitrogen. vsd files using Microsoft Visio 2016 Viewer 64 bit. GEO help: Mouse over screen elements for information.
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