Gene expression profiling of murine BCR-ABL+ CDK6-wt and CDK6-KO cells

Dertermination of chromatin accessibility in murine BCR-ABL+ CDK6-wt and CDK6-KO cells Overall design: RNA-seq:Murine (C57Bl/6J) CDK6wt and CDK6-/- cell lines obtained from single cell bone marrow suspension were retrovirally transduced with a pMSCV-BCR-ABLp185-IRES-GFP vector. To test for differences in transcriptional gene expression RNA from CDK6wt and CDK6-/- cell lines was analysed by RNA-seq. Thus, total RNA was prepared using the RNeasy Kit (Qiagen) and processed for sequencing using the TruSeq RNA Sample Preparation Kit (Illumina Inc, San Diego, CA, USA). Sequencing was performed using the Illumina HiSeq3000/4000 platform. RNA-seq experiments were performed in biological triplicates. ATAC-seq: Murine (C57Bl/6J) CDK6wt and CDK6-/- cell lines obtained from single cell bone marrow suspension were retrovirally transduced with a pMSCV-BCR-ABLp185-IRES-GFP vector. Chromatin accessibility was assessed by ATAC-seq. Briefly, cells were washed once in 50?µl PBS and resuspended in transposase reaction mix (12.5?µl 2 × TD buffer, 2?µl transposase (Illumina), 10.5?µl nuclease-free water and 0.01% NP-40). Tagmentation was performed for 30?min at 37?°C. Following library amplification, fragments larger than 1,200?bp were excluded by SPRI size selection. Libraries were amplified using custom Nextera primers and sequenced using the Illumina HiSeq3000/4000 platform.