Distinct Immunological Features Compared to Lichen Planus and Oral Lichen Planus

Description

Single Cell 3' libraries were prepared using standard Illumina paired-end constructs, which include the P5 and P7 adaptors. The libraries incorporated a 16 bp 10x Barcode and a 12 bp Unique Molecular Identifier (UMI) within Read 1. The cDNA fragments in the 3' Gene Expression libraries were sequenced using Read 2, while the DNA from Cell Multiplexing Oligo Feature Barcodes in the Cell Multiplexing libraries was sequenced using Read 2N. The libraries were subjected to paired-end sequencing on an Illumina platform. Sample indexing was achieved using i7 and i5 index sequences. The following standard Illumina sequencing primer sites were utilized: for the 3' Gene Expression libraries, TruSeq Read 1 and TruSeq Read 2 primers were employed; for the Cell Multiplexing libraries, Nextera Read 1 and Nextera Read 2 primers were used. This method ensured accurate and efficient sequencing of both gene expression and multiplexing features, facilitating comprehensive single-cell analysis.

Steps to reproduce

We assessed the quality and basic statistics of the raw sequencing data using FastQC. The data were processed with Cell Ranger v6.0.2 using the human genome (GRCh38). We filtered out low-quality UMIs, keeping cells with 500-6000 detected genes and less than 5% mitochondrial reads. Samples were normalized with log normalization, and the top 2000 highly variable genes were identified. Dimensionality reduction was performed using PCA, t-SNE, and UMAP. For annotation, we mapped our data to a PBMC reference dataset using Seurat V4 and SingleR.

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Licence