NextSeq 500 paired end sequencing; CSS-mRNA_A1-Jax11_M3_PP_H3GJTBGX3_lane1
Protocols :  Extraction Protocol: RNA was extracted from 10^5-10^6 sorted cells using 1000 microliter of TRI-Reagent (Sigma). Cell lysate were added to the tubes containing pre-spun Phase Lock Gel-Heavy and incubated for 5 minutes at room temperature. 200 microliter of chloroform per 1000 microliter of trizol were added to the tube and centrifuged at 12 000 x g at 4C for 10 minutes. The clear aqueous phase was collected and mixed with equal volume of 100% isopropanol. The resulting solution was loaded on Qiagen RNA columns. On column DNase treatment was performed using RNase-free DNase Set. RNA columns were washed twice with wash solution, and total RNA was eluted in Molecular Biology Grade water. RNA was quantified using the NanoDrop 1000 spectrophotometer (Thermo Scientific). RNA integrity was analysed using the Bioanalyzer RNA Nano Kit (Agilent). Library Protocol: Barcoded stranded mRNA-seq libraries were prepared from high quality total RNA samples (~200 ng/sample, RIN>9) using the Illumina TruSeq RNA Sample Preparation v2 Kit (Illumina, San Diego, CA, USA) implemented on the liquid handling robot Beckman FXP2 following the manufacturerâ€™s recommendations. Library quality was assessed on the Bioanlyzer 2100 using DNA High Sensitivity kit. The library size distribution was determined using Agilent 2100 expert software.
Factor Value[Cell Type]
PrePro B-cells
Factor Value[Strain / Line]
C57BL/6J