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 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.

本数据集针对小鼠BCR-ABL阳性、CDK6野生型（CDK6-wt）与CDK6敲除（CDK6-KO）细胞的染色质开放状态及转录组特征开展研究，包含RNA测序（RNA-seq）与转座酶可及性测序（ATAC-seq）两类高通量测序数据，具体实验流程如下：

RNA-seq实验：从单细胞骨髓悬液中分离得到的C57Bl/6J小鼠CDK6野生型与CDK6敲除细胞系，经逆转录病毒转导pMSCV-BCR-ABLp185-IRES-GFP载体。为探究转录基因表达的差异，对两组细胞系的RNA样本进行RNA-seq分析。总RNA采用RNeasy试剂盒（Qiagen公司）提取，文库构建使用TruSeq RNA样本制备试剂盒（美国加利福尼亚州圣地亚哥Illumina公司），测序在Illumina HiSeq3000/4000平台完成，实验设置三次生物学重复。

ATAC-seq实验：同样使用上述经逆转录病毒转导的C57Bl/6J小鼠CDK6野生型与CDK6敲除细胞系，通过ATAC-seq检测染色质开放状态。具体实验步骤如下：细胞经50μL磷酸盐缓冲液（PBS）洗涤一次后，重悬于转座酶反应体系（含12.5μL 2×TD缓冲液、2μL转座酶（Illumina公司）、10.5μL无酶水及0.01% NP-40）中，37℃下孵育30分钟完成转座酶切割标记。文库扩增后，通过SPRI磁珠大小筛选排除大于1200bp的片段；随后使用定制Nextera引物对文库进行扩增，最终在Illumina HiSeq3000/4000平台完成测序。