title: GSM4017199: CD44pos; Homo sapiens; RNA-Seq
library_construction_protocol: The cells were detached from the flask with 7 mls of 1X citrate saline to which 7mls of DPBS was added followed by centrifugation at 300xG for 7 minutes. The cells pellet was resuspended in 300 ul of blocking buffer (Rat IgG/PBS) and incubated on ice for 30 minutes. 50ul of the cells in blocking buffer were transferred to a separate tube for the isotype control. The cells were washed with1ml of staining buffer (5% BSA/PBS) and centrifuged at 300XG for 5 minutes. The pellet containing approximately 3X10^6 cells was incubated with Rat IgG2B anti-CD44-Alexa 488 antibody (R&D systems) in staining buffer (15ul antibody + 135 ul of staining buffer) for 30 minutes on ice. For the isotype control ~600,000 cells were incubated with 5ul of Rat IgG2B-Alexa488 (R&D systems) + 45ul of staining buffer for 30 minutes on ices. After the incubation both sets of cells were pelleted and washed with 1ml of staining buffer as described above and resuspended in staining buffer, followed by flow cytometric analysis to identify the fraction of CD44 positive and negative populations. For the single cell experiment Rh41 cells were cultured and harvested and washed in DPBS as described above, and resuspended in PBS/0.2%BSA at a concentration of 1X106 cells per ml. The 10x Genomics single-cell platform performs 3' gene expression profiling by poly-A selection of mRNA within a single cell, which utilizes a cell barcode and UMIs (unique molecular identifiers) for each transcript. Single-cell suspensions were loaded onto the Chromium Controller according to their respective cell counts to generate approximately 6000 partitioned single-cell GEMs (Gel Bead-In-EMulsions). The library was prepared using the Chromium Single Cell 3' v2 Library and Gel Bead Kit (10x Genomics) according to the manufacturers protocol. The cDNA content of each sample after cDNA amplification of 12 cycles was quantified and quality checked by High-Sensitivity DNA chip on a 2100 Bioanalyzer (Agilent Technologies) at a dilution of 1:6. This quantification was used to determine final library amplification cycles in the protocol, which calculated out to 12 cycles. After library quantification and quality check by DNA 1000 chip (Agilent Technologies), samples were diluted to 3.5nM for loading onto the HiSeq 4000 (Illumina) with a 2 x 75 Paired-end kit using the following read length: 26 bp Read1 (10x cell barcode and UMI), 8 bp i7 Index (sample index), and 98 bp Read2 (insert). An average of 400,000,000 reads per sample was obtained, which translated to roughly 80,000 mean reads per cell, per sample.
attributes.tag: GEO Accession
attributes.value: GSM4017199