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Single cell transcriptome map of human bone marrow hematopoietic stem/progenitor cells and construction method

A technology for bone marrow hematopoiesis and progenitor cells, which can be applied in the fields of instruments, analytical materials, computer parts, etc.

Pending Publication Date: 2022-03-15
细胞生态海河实验室 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these studies have greatly improved people's understanding of hematopoiesis, the depth of sequencing and the acquisition of less information limit the understanding

Method used

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  • Single cell transcriptome map of human bone marrow hematopoietic stem/progenitor cells and construction method
  • Single cell transcriptome map of human bone marrow hematopoietic stem/progenitor cells and construction method
  • Single cell transcriptome map of human bone marrow hematopoietic stem/progenitor cells and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] The present embodiment sorts human HSPC cells, comprising the following steps:

[0069] (1) collecting human bone marrow cells and extracting mononuclear cells;

[0070] (2) Resuspend cells in 1 mL of IMDM medium containing 20% ​​FBS and 100 μg / mL DNAse I, mix well and place at 25°C for 13 minutes to remove clumps released by dead cells;

[0071] (3) Add 2 mL of IMDM containing 10% FBS, centrifuge at 4°C and 1000 rpm for 20 min, discard the supernatant; every 10 7 Cells were labeled with antibodies as shown in Table 1, and protected from light at 4°C for 30 minutes;

[0072] (4) Add 1 mL of PBS buffer, centrifuge at 1500 rpm for 5 min at 4°C, and resuspend the cells in PBS buffer (1 mL / 10 7 cells), adding DAPI at a ratio of 1:1000 for sorting.

[0073] Table 1

[0074]

[0075]

[0076] Sorting results such as figure 1 As shown, there are 8 groups of cells including HSC, MPP, LMPP, MLP, CMP, GMP, MEP and B-NK. The flow cytometric markers of HSC, MPP, LMPP, ML...

Embodiment 2

[0086] This embodiment constructs the HSPC single-cell transcriptome library, including the following steps:

[0087] (1) Single cell lysis:

[0088] a. According to Table 2, prepare the lysate in eight consecutive tubes and store at -80°C;

[0089] Table 2

[0090]

[0091]

[0092] b. placing the single HPSC cell sorted in Example 1 into eight consecutive tubes;

[0093] c. Adjust the vortex instrument to the maximum speed of 10, vortex the eight-row row for 45s (4 eight-row rows at a time), centrifuge instantaneously, put it in the PCR instrument for 3min (72°C), take it out and put it on ice;

[0094] (2) Single-cell RNA reverse transcription

[0095] a. prepare the reverse transcription reaction system according to Table 3;

[0096] table 3

[0097] components Volume (μL) SuperScript II reverse transcriptase (200U / μL) 0.25 RNAse inhibitor (40U / μL) 0.125 Superscript II first-strand buffer(5x) 1 0.1M DTT 0.25 Betaine (5M) ...

Embodiment 3

[0161] In this example, the HPSC single-cell transcriptome library constructed in Example 2 was sequenced.

[0162] Perform STRT-seq single-cell transcriptome sequencing (sequencing completed by Novogene).

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Abstract

The invention discloses a single cell transcriptome map of human bone marrow hematopoietic stem / progenitor cells and a construction method. The method comprises the following steps: (1) sorting human bone marrow hematopoietic stem / progenitor cells; (2) constructing a single cell transcriptome library of the human bone marrow hematopoietic stem / progenitor cells; (3) sequencing the single cell transcriptome library; (4) analyzing the sequenced data, and constructing a single cell transcriptome map of the human bone marrow hematopoietic stem / progenitor cells; through deep single cell transcriptome sequencing, the single cell transcriptome map of the human bone marrow hematopoietic stem / progenitor cells is constructed to systematically research the characteristics of the human hematopoietic stem / progenitor cells, more information is obtained, and a theoretical basis can be provided for exploring physiological and pathological hematopoiesis of the human bone marrow hematopoietic stem / progenitor cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a single-cell transcriptome map and a construction method of human bone marrow hematopoietic stem / progenitor cells. Background technique [0002] Hematopoietic stem cells (HSCs) are the most studied adult stem cells in the body. HSCs are rare and usually exist in the bone marrow microenvironment in a resting state, and are strictly regulated by various external factors and intracellular molecules in the microenvironment. . As the "seed" cell of the entire hematopoietic system, it is located at the apex of the differentiation and development of the hematopoietic system. The traditional view is that there is an obvious hierarchical relationship between HSC and its progeny cells and mature blood cells, that is, HSC generates multipotential progenitor cells (MPP), and then differentiates into common lymphoid progenitor cells (CLP) and common myeloid progenitor cells. Cells (CMP), and the...

Claims

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Application Information

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IPC IPC(8): G16B25/10G16B30/10G16B40/00G16B45/00G01N15/14G06K9/62
CPCG16B25/10G16B30/10G16B40/00G16B45/00G01N15/1404G06F18/23G06F18/213G06F18/2135G06F18/24
Inventor 程涛胡林萍朱平谢小韦张雅文尹秀秀
Owner 细胞生态海河实验室
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