GSM5971435: Patient_1217_PreTreatment CNV; Homo sapiens; OTHER
For single-cell DNA library generation for CNV single-cell suspensions of primary MM cells were processed according to 10X Genomics Reagent Kits User Guide (CG000153). Single cells were partitioned in a hydrogel matrix by combining with a CB polymer to form Cell beads (CBs) using a microfluidic chip. Post a first encapsulation, CBs were treated to lyse the encapsulated cells and denature the genomic DNA (gDNA). The denatured gDNAs in the CB were then accessible to amplification and barcoding. A second microfluidic encapsulation step was then required to partition the CB with 10x barcode Gel Beads (GBs) to generate GEMs. Immediately after barcoding and amplification, 10x barcoded fragments were pooled and attached to standard Illumina adaptors. For all the single cell methods, quality control and quantification was performed using a Kapa Library Quantification qPCR kit (Kapa Biosystems) on a BioRad qPCR instrument prior to preparing a single pool containing equal amounts of each library. This pool was then subjected to on-board cluster formation and sequencing on an Illumina NextSeq 500 sequencer with a high-output v2.5 150 sequencing kit for RNA-seq and ATAC-seq and 300 cycle sequencing kit for CNV-seq as per the standard Illumina protocols. After sequencing, the bcl data was converted to fastq data files using the Illumina BCL2FASTQ utility. Samples were processed with CellRanger suite v3.0 and downstream analyses were realized with Seurat, Monocle, Signac, and Cicero R packages.