title: GSM8186644: Kabuki syndrome type 1 replicate 4; Mus musculus; RNA-Seq
library_name: GSM8186644
library_construction_protocol: T cells were isolated at exactly the same time as B cells. Specifically, flow through from binding and washing of CD19+ cells on Miltenyi separation columns was collected on ice. These CD19+ depleted samples were then spun and the supernatant was aspirated, resuspended and incubated with 2x (20uL) CD90.2+ microbeads (Miltenyi Biotec 130-121-278). CD90.2+ T cells were bound, washed and eluted per manufacturer protocol, then counted and aliquoted on ice for subsequent processing. Further processing of the T cell samples and RNA sequencing was done in parallel with B cells. Specifically, 100–500k cells were spun at 300–500 g for 5 min at 4', were homogenized in Trizol (Invitrogen 15596018) and subsequently stored at –80°C until RNA was extracted. We extracted and isolated RNA by phase separation with standard protocols, with purification using the Direct-zol RNA microprep kit (Zymo R2060) with an on-column DNAse step as per manufacturer's directions. Following purification, RNA was quantified with the Quant-iT RiboGreen RNA Assay Kit (ThermoFisher R11490) or Qubit RNA HS Assay Kit (ThermoFisher Q32852). We assessed the quality of the RNA using Bioanalyzer with the RNA 6000 Pico Kit (Agilent 5067–1513). We used 20 ng RNA per sample were used as input to capture mRNA (NEBNext Poly(A) mRNA Magnetic Isolation Module; NEB #E7490), and subsequently libraries were generated with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760/E7765) as per manufacturer's protocols. Library size and quality were then evaluated with BioAnalyzer with DNA High Sensitivity reagents (Agilent 5067–4626), and concentration was assessed using the Qubit dsDNA HS Assay Kit (ThermoFisher Q32851) and KAPA Library Quantification Kit for qPCR (KAPA KK4824). Finally, samples were pooled and sequenced on Illumina HiSeq4000 using PE flow cells with 100-8-8-100 read length as per standard manufacturer's protocols. Libraries were generated using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760/E7765) per manufacturer protocols. Library size and quality were determined using BioAnalyzer with DNA High Sensitivity reagents (Agilent 5067-4626), and concentration was determined using Qubit dsDNA HS Assay Kit (ThermoFisher Q32851) and KAPA Library Quantification Kit for qPCR (KAPA KK4824).