GSM1897409: pro-B YY1 KO RNA-seq_r2; Mus musculus; RNA-Seq
For bone marrow subset purification we used B6.RAG1-/-mice to obtain pro-B cells. Pro-B YY1 KO was obtained with RAG-/-YY1f/f x mb1-Cre pro-B cells. B6.RAG-/-µ+ mice bearing a rearranged IgM transgene were used to obtain pre-B cells. follicular B cells were sorted from 5-8 week old spleen as CD19+AA4- CD21loCD23hi. Germinal center B cells were obtained from spleens of mice 8 days after IP immunization with 100 mg OVA and 10 mg of LPS in alum.  GC B cells were identified as CD19+Fas+GL7+ Directional RNASeq. For each sample approximately 0.5 ug total RNA was depleted of ribosomal RNA using RiboMinus (Life Technologies). Ribodepleted RNA was prepared for sequencing using the NEBNext Ultra RNA Library Prep Kit (New England Biolabs) for Illumina following manufacturer’s recommend protocols with 15 cycles of PCR. Final libraries were size selected on 2% agarose gels to obtain products with library sizes between 280-380 bases in length. The prepared libraries were loaded onto an Illumina HiSeq2000 for single-end 100 base reads with 7 bases of the index read. Data was processed to generate Fastq files using CASAVA 1.8 and demultiplexed based on index sequences.