alias: GSM1565359
title: GSM1565359: Small cell from well C12; Mus musculus; RNA-Seq
library_construction_protocol: Primary splenocytes were isolated from 6-8 week old mice, naïve B cells purified using magnetic bead separation (Miltenyi Biotec), and stimulated with 250 ng/mL CpG ODN 1668 (Invivogen). B cells were grown in fresh media with 1% penicillin streptomycin solution (Mediatech Inc.), 5 mM L-glutamine (Mediatech Inc.), 25 mM HEPES buffer (Mediatech Inc.), 10% FCS and 55 uM 2-ME (Fisher Scientific) at a concentration of 5x10^4 cells/mL in 48 well plates at 37oC for a 24 h period. Stimulated wildtype B cells were collected at 24 h post stimulation and concentrated to 5x10^5 cells per mL. Cells were loaded onto a 10-17 uM primed C1 single-cell auto prep array IFC (Fluidigm), and phase contrast images were taken of all viable cells as determined by the Live/Dead® Viability/Cytotoxicity Kit (Invitrogen). ERCC RNA spikein controls (LifeTechnologies) were added to the lysis mix at a 1:200 dilution. Tube controls (bulk cell positive control, and no cell negative control) were also prepared according to the Fluidigm protocol. Lysis, reverse transcription, and PCR were performed using the SMARTer Ultra Low RNA Kit (CloneTech) and Advantage® 2 PCR Kit (CloneTech) on individual cells using the C1 Single-Cell Auto Prep System (Fluidigm). Cell size was manually determined from images using ImageJ software. Sample libraries for the five smallest and five largest cells along with the controls were prepared using the Nextera XT DNA Sample Preparation (Illumina), and library quality was assayed using the Quant-iT PicoGreen dye (Life Technologies) quantification on a Qubit® 2.0 Fluorometer (LifeTechnologies) and by gel electrophoresis.
identifiers.namespace: GEO
identifiers.id: GSM1565359
attributes.tag: GEO Accession
attributes.value: GSM1565359