GSM3176108: PD11; Mus musculus; RNA-Seq
Single cell suspensions were obtained total splenocytes  in aged (6Mo) mice. Single cell suspensions from spleens were placed at 4C and then surface stained in FACS buffer (1X PBS with 3% BSA) containing fluorochrome-conjugated monoclonal antibodies and then stained with a fixable viability dye prior to fluorescence activated cell sorting (FACs). Germinal Center B cells (GC B cells) were defined as Live/Dead-neg, B220-pos, GL7-pos and CD95-pos, whereas non-germinal center B cells (Non-GC B cells) were defined as Live/Dead-neg, B220-pos, GL7-neg, CD95-neg.  All FACS-sorting was performed using a FACSAria (BD) High Speed cell sorter. Cells were sorted directly into Trizol reagent to isolate RNA for further RNA-sequencing (RNA-seq).   RNA was phase separated using chloroform and the aqueous phase containing RNA was removed following centrifugation and precipitated overnight at -20C using ice cold isopropanol. RNA was washed with 75% ethanol then solubilized in RNase-free water. Optical density values of extracted RNA were measured using NanoDrop (Thermo Fisher Scientific) to confirm an A260:A280 ratio above 1.9. RNA integrity number (RIN) was measured using BioAnalyzer (Agilent) RNA 6000 Pico Kit to confirm RIN above 7. The cDNA libraries were prepared using the SMARTer® Ultra® Low Input RNA Kit for Sequencing - v3 (Clontech) followed by Nextera XT DNA Library Prep Kit (Illumina) as per the manufacturer's instructions. The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). Single cell suspensions were obtained total splenocytes  in aged (6Mo) mice. Single cell suspensions from spleens were placed at 4C and then surface stained in FACS buffer (1X PBS with 3% BSA) containing fluorochrome-conjugated monoclonal antibodies and then stained with a fixable viability dye prior to fluorescence activated cell sorting (FACs). Germinal Center B cells (GC B cells) were defined as Live/Dead-neg, B220-pos, GL7-pos and CD95-pos, whereas non-germinal center B cells (Non-GC B cells) were defined as Live/Dead-neg, B220-pos, GL7-neg, CD95-neg.  All FACS-sorting was performed using a FACSAria (BD) High Speed cell sorter. Cells were sorted directly into Trizol reagent to isolate RNA for further RNA-sequencing (RNA-seq).   RNA was phase separated using chloroform and the aqueous phase containing RNA was removed following centrifugation and precipitated overnight at -20C using ice cold isopropanol. RNA was washed with 75% ethanol then solubilized in RNase-free water. Optical density values of extracted RNA were measured using NanoDrop (Thermo Fisher Scientific) to confirm an A260:A280 ratio above 1.9. RNA integrity number (RIN) was measured using BioAnalyzer (Agilent) RNA 6000 Pico Kit to confirm RIN above 7. The cDNA libraries were prepared using the SMARTer® Ultra® Low Input RNA Kit for Sequencing - v3 (Clontech) followed by Nextera XT DNA Library Prep Kit (Illumina) as per the manufacturer's instructions. The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). Single cell suspensions were obtained total splenocytes  in aged (6Mo) mice. Single cell suspensions from spleens were placed at 4C and then surface stained in FACS buffer (1X PBS with 3% BSA) containing fluorochrome-conjugated monoclonal antibodies and then stained with a fixable viability dye prior to fluorescence activated cell sorting (FACs). Germinal Center B cells (GC B cells) were defined as Live/Dead-neg, B220-pos, GL7-pos and CD95-pos, whereas non-germinal center B cells (Non-GC B cells) were defined as Live/Dead-neg, B220-pos, GL7-neg, CD95-neg.  All FACS-sorting was performed using a FACSAria (BD) High Speed cell sorter. Cells were sorted directly into Trizol reagent to isolate RNA for further RNA-sequencing (RNA-seq).   RNA was phase separated using chloroform and the aqueous phase containing RNA was removed following centrifugation and precipitated overnight at -20C using ice cold isopropanol. RNA was washed with 75% ethanol then solubilized in RNase-free water. Optical density values of extracted RNA were measured using NanoDrop (Thermo Fisher Scientific) to confirm an A260:A280 ratio above 1.9. RNA integrity number (RIN) was measured using BioAnalyzer (Agilent) RNA 6000 Pico Kit to confirm RIN above 7. The cDNA libraries were prepared using the Ovation RNA-seq System V2 (NuGEN, San Carlos, CA) as per the manufacturer's instructions.  The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies) and then loaded onto TruSeq SR v3 flow cells on an Illumina HiSeq 2500 (Illumina, San Diego, CA) and run for 100 cycles using a paired-read recipe (TruSeq SBS Kit v3, Illumina) according to the manufacturer's instructions.
GEO Accession
GSM3176108