title: WT_3_ProB
organism: Mus musculus
description: Protocols: Single cell suspensions generated from BM were stained with antibodies against CD19, IgM, CD43 and B220. Dead cells were stained by addition of 7AAD. For each sample, 50 Pro B cells were sorted into 4 wells of a 96 well plate (200 cells in total per sample) based on 7AAD-,CD43+IgM-CD19+B220+. In addition, from mutant mice 7AAD-,CD43+CD24+IgM-CD19+B220- cells were also sorted. RNA was extracted using a modified version of the SMARTseq2 protocol from Picelli et al., 2014. 50 Cells were sorted into 96 well plates (4 wells per sample) containing 1ul of the cell lysis buffer, 0.5mL dNTP mix (10mM) and 0.5mL of the oligo dT primer at 5mM. We then reduced the amount of reagent used in the following reverse transcription and PCR amplification step by half. The concentration of the IS PCR primer was also further reduced to 50nM. Due to the low transcriptional activity of resting B cells, we increased the number of PCR cycles to 28. Sequencing libraries were prepared using the Nextera XT Library Preparation Kit with the protocol modified by reducing the original volumes of all reagents in the kit by 1/5th.