Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes.
Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes. Mouse hematopoietic stem cells were purified from bone marrow cells using negative and positive selection with a Magnetic-Activated Cell Sorter (MACS). total RNA and mRNA were purified from the purified cells using Trizol reagent and magnetic oligo dT beads. Double strand cDNAs were synthesized using a cDNA synthesis kit and anchored oligo dT primers. After NlaIII digestion, 3’ cDNAs were isolated and amplified through 16-cycle PCR. SAGE tags were released from the 3’ cDNA after linker ligation. Ditags were formed, concatemerized and cloned into a pZERO vector. Sequencing reactions were performed with the ET sequencing terminator kit. Sequences were collected using a Megabase 1000 sequencer. SAGE tag sequences were extracted using SAGE 2000 software.
Project description:A single hematopoietic stem cell can give rise to all blood cells with remarkable fidelity. Here, we define the chromatin accessibility and transcriptional landscape controlling this process in thirteen primary cell types that traverse the hematopoietic hierarchy. Exploiting the finding that enhancer landscapes better reflect cell identity than mRNA levels, we enable "enhancer cytometry" for accurate enumeration of pure cell types from complex populations. We further reveal the lineage ontogeny of genetic elements linked to diverse human diseases. In acute myeloid leukemia, chromatin accessibility reveals distinctive regulatory evolution in pre-leukemic HSCs (pHSCs), leukemia stem cells, and leukemic blasts. These leukemic cells demonstrate unique lineage infidelity, confirmed by single cell regulomes. We further show that pHSCs have a competitive advantage that is conferred by reduced chromatin accessibility at HOXA9 targets and is associated with adverse patient outcomes. Thus, regulome dynamics can provide diverse insights into human hematopoietic development and disease. Single-cell ATAC-seq of LMPPs, Monocytes, LSCs and Luekemic blast cells.
Project description:A single hematopoietic stem cell can give rise to all blood cells with remarkable fidelity. Here, we define the chromatin accessibility and transcriptional landscape controlling this process in thirteen primary cell types that traverse the hematopoietic hierarchy. Exploiting the finding that enhancer landscapes better reflect cell identity than mRNA levels, we enable "enhancer cytometry" for accurate enumeration of pure cell types from complex populations. We further reveal the lineage ontogeny of genetic elements linked to diverse human diseases. In acute myeloid leukemia, chromatin accessibility reveals distinctive regulatory evolution in pre-leukemic HSCs (pHSCs), leukemia stem cells, and leukemic blasts. These leukemic cells demonstrate unique lineage infidelity, confirmed by single cell regulomes. We further show that pHSCs have a competitive advantage that is conferred by reduced chromatin accessibility at HOXA9 targets and is associated with adverse patient outcomes. Thus, regulome dynamics can provide diverse insights into human hematopoietic development and disease. ATAC-seq profiles of hematopoietic and leukemic cell types, across 13 normal hematopoietic cell types and 3 acute myeloid leukemia cell types. The complete data set contains a total of 132 samples.