snRNA-seq, Primary-Recurrent GBM (Mikolajewicz Cohort)

<b>Summary.</b><br>10 primary GBM and 8 recurrent GBM samples (14/18 matched) profiled using single nucleus RNA- sequencing (sci-RNA-seq3 protocol).<br><b>Data Format.</b><br>Data is provided as preprocessed dataset, stored in Seurat Object.<br><b>Sample processing, sci-RNA-seq3 library generation, and sequencing</b><br>Snap-frozen patient pGBM and rGBM tissues were chopped with a razor blade or scissors before nucleus isolation. Nuclei extraction and fixation were performed as previously described (Cao 2019), except for the use of a modified CST lysis buffer50 plus 1% of SUPERase-In RNase Inhibitor (Invitrogen, #AM2696). Lysis time and washing steps were further optimized based on human GBM tissue. Nuclei quality was checked with DAPI and Wheat Germ Agglutinin (WGA) staining. Sci-RNA-seq3 libraries were generated as previously described49 using three-level combinatorial indexing. The final libraries were sequenced on Illumina NovaSeq as follows: read 1: 34bp, read 2: &gt;=69bp, index 1: 10bp, index 2: 10bp.<br><b>Demultiplexing and read alignments.</b><br>Raw sequencing reads were first demultiplexed based on i5/i7 PCR barcodes. FASTQ files were then processed using the sci-RNA-Seq3 pipeline. After barcodes and unique molecular identifiers (UMIs) were extracted from the read1 of FASTQ files, read alignment was performed using STAR short-read aligner (v2.5.2b) with the human genome (hg19) and Gencode v24 gene annotations. After removing duplicate reads based on UMI, barcode, chromosome and alignment position, reads were summarized into a count matrix of M genes × N nuclei.<br><b>Filtering, normalization, integration, and dimensional reduction.</b><br>Raw count matrices were loaded into a Seurat object (version 4.0.1) and filtered to retain cells with (i) 200 – 9000 recovered genes per cell, (ii) less than 60% mitochondrial content, and (iii) unmatched rate within 3 median absolute deviations of the median. To normalize count matrix, we adopted the modeling framework previously described and implemented in sctransform (R Package, version 0.3.2). In brief, count data were modelled by regularized negative binomial regression, using sequencing depth as a model covariate to regress out the influence of technical effects, and Pearson residuals were used as the normalized and variance stabilized biological signal for downstream analysis. Data from each patient were integrated with the reciprocal PCA method (Seurat) using the top 2000 variable features. PCA was performed on the integrated dataset, and the top N components that accounted for 90% of the observed variance were used for UMAP embedding, RunUMAP(max_components = 2, n_neighbours = 50, min_dist = 01, metric = cosine).<br><b>Contact.</b><br>Contact Dr. Nicholas Mikolajewicz regarding any questions about the data or analysis ([e-mail])