RNASeq, multiome, and genomic profiling of hematopoietic progenitors and B cells from mice with a point mutation in MYC [Multiome]. RNASeq, multiome, and genomic profiling of hematopoietic progenitors and B cells from mice with a point mutation in MYC [Multiome]

We generated mice with a mutation at threonine 58 of MYC in the mouse germline (T58A mutant). These mice ultimately develop AML or B cell lymphomas. We profiled changes in gene expression and genomic occupation of MYC in single cells of sorted, immature bone marrow progenitor cells, mature (spleen derived) and immature (marrow derived) B cells. B cells were stimulated with LPS and IL7, respectively. Overall design: Lineage negative, ckit+, Sca1+ cells were sorted from marrow of WT or T58A littermate mice, and profiled for expression and DNA accessibility by single cell RNA-Seq and ATAC-Seq using the 10X genomic system. Bulk RNA-Seq was also performed on immature B cells isolated from bone marrow, and stimulated with IL7. RNA-Seq was also done on mature B cells isolated from spleens of these animals, and stimulated with lipopolysaccharide (LPS). Genomic profiling was done using CUT&RUN to determine MYC occupation, and histone modifications (H3K27Ac and H3K4Me2) were carried out in both the LPS and IL7 treated cells. For scRNA-Seq, 2 paired, biological replicate experiments (LSK_1 and LSK_2, comparing sorted cells from WT and T58A mutant mice) were done for each genotype. Each replicate consisted of LSK cells from 2 mice of each genotype, for a total of 4 mice per genotype for this experiment. For all genomic profiling experiments (CUT&RUN), 2 biological replicates per genoype (mice) were performed.

我们在小鼠生殖系中引入MYC基因苏氨酸58位点突变（T58A突变体），构建了该突变小鼠模型。此类小鼠最终可罹患急性髓系白血病（AML）或B细胞淋巴瘤。
我们对分选获得的未成熟骨髓祖细胞、成熟（脾脏来源）与未成熟（骨髓来源）B细胞的单细胞进行了基因表达谱与MYC基因组结合占据情况的分析；其中B细胞分别经脂多糖（LPS）与白细胞介素7（IL7）刺激。
整体实验设计如下：从野生型（WT）与T58A突变型同窝小鼠的骨髓中分选谱系阴性、ckit阳性、Sca1阳性（LSK）细胞，借助10X Genomics平台，通过单细胞RNA测序（scRNA-Seq）与转座酶可及性测序（ATAC-Seq）分析其基因表达与DNA染色质可及性。
我们还对从骨髓中分离并经IL7刺激的未成熟B细胞开展批量RNA测序（bulk RNA-Seq）；同时对从这些动物脾脏中分离并经脂多糖（LPS）刺激的成熟B细胞进行RNA测序。
基因组谱分析采用CUT&RUN技术检测MYC的基因组结合占据情况，并分别在LPS与IL7刺激的细胞中检测组蛋白修饰：H3K27乙酰化（H3K27Ac）与H3K4二甲基化（H3K4Me2）。
针对单细胞RNA测序实验，每种基因型设置2组配对生物学重复实验（LSK_1与LSK_2，比较野生型与T58A突变型小鼠的分选细胞）；每个重复使用2只同基因型小鼠的LSK细胞，因此该实验每组基因型共使用4只小鼠。
所有基因组分析实验（CUT&RUN）每组基因型均设置2只生物学重复小鼠。