GSM8019069: lung sample, IPF patient, scRNA-seq; Homo sapiens; RNA-Seq
The lung sample (n=1) from IPF patient was harvested and dissociated into single cells suspensions by mixed digestion buffer. Cells were collected after filtering through a 70 μm and 40 μm strainer. The viability and concentration of cells were calculated in a Counting Star system. Library was performed according to the manufacter's instructions (single cell 5' protocol, 10x Genomics). Briefly, GEMs (Gel Bead-in-Emulsions) are generated using Single Cell 5' Gel Beads, Master Mix, cells, and Partitioning Oil on a microfluidic chip to achieve single cell resolution. The majority of GEMs are cell-free, while a minority contain a single cell. Upon GEM formation, Gel Bead dissolution releases oligonucleotides including Illumina® R1 sequence, 10x Barcode, Unique Molecular Identifier (UMI), and Switch Oligo into the cell lysate, followed by reverse transcription to create barcoded cDNA from mRNA. Post GEM-RT, Silane magnetic beads purify barcoded cDNA, which is then amplified via PCR for library construction. cDNA amplification is performed with PCR primers against common 5' and 3' ends, preparing for multiple library types, including T/B cell-enriched and 5' gene expression libraries. For target enrichment, barcoded V(D)J segments from amplified cDNA are enriched via PCR using TCR or Ig constant region-specific primers, with separate reactions for T and B cells if both are present. Enriched library construction involves Enzymatic Fragmentation and Size Selection for V(D)J segments, incorporating sequencing elements like R1, P5, P7, R2, and sample index during different stages, producing final libraries compatible with Illumina® bridge amplification. The Single Cell V(D)J Reagent Kit is used for Illumina-ready V(D)J enriched and 5' gene expression sequencing libraries, which have standard Illumina paired-end constructs encoding barcode and primer sequences, as well as sample indices.