Welcome to Axolotl Cell Atlas

Ambystoma mexicanum scRNA-seq

Axolotl Cell Atlas View in gallery

CH-seq protocol

Combinatorial hybridization sequencing (CH-seq) is a flexible pool-split based high-throughput (10,000-4 million cells/nuclei per experiment) single cell/nucleus library preparation method. This strategy could apply to single cell/nucleus RNA sequencing, single nucleus DNA sequencing (chromatin accessibility) as well as other omics.

Work flow

1.Single cells/nuclei preparation.
2.Cells/nuclei fixation by paraformaldehyde/formaldehyde
3.Membrane permeabilization, filtering and counting.
4.Reverse transcription with 1st barcoded polyT oligos (CH-RNA-seq) or tagmentation with 1st barcoded transposase (CH-ATAC-seq) into one or more 96-well plates. Pool and randomly redistribute cells/nuclei into one or more 96-well plates.
5.1st polyT barcode oligos (CH-RNA-seq) or transposase adaptor oligos (CH-ATAC-seq) hybridized with 2nd barcoded annealing oligos without ligase in hybridization buffer.
6.Block extra free annealing hybridization oligos. Pool and randomly redistribute cells/nuclei into one or more 96-well plates.
7.In-cells/nuclei second strand synthesis (CH-RNA-seq) or gap filling (CH-ATAC-seq). Lysis, purification, tagmentation and indexed PCR (CH-RNA-seq) or directly indexed PCR (CH-ATAC-seq).
8.Library purification and sequencing.

Materials and equipment

Oligos and Reagents

1. 384 barcoded oligodT primers, 384 barcoded transposase adaptor primers, 768 barcoded hybridization annealing primers, block oligo, 96 index PCR primers (see below Table S1).
2. DNase/RNase free water.
3. DEPC Treated Water.
4. DNase/RNase free pipet tips.
5. Cell strainers (20um, 40um).
6. Paraformaldehyde/formaldehyde aqueous solutions.
7. 1X phosphate buffered saline (PBS), 1X dulbecco's modified eagle medium (DMEM)
8. 1M Tris-HCL (pH 7.5)
9. 3M NaCl
10. 1M MgCl2
11. IGPAL-CA 630
12. Tween-20
13. Glycerol.
14. Mg (OAc)2
15. Polyethylene glycol (PEG 8000).
16. 0.5M Ethylene Diamine Tetraacetic Acid (EDTA).
17. HCl solution.
18. Dithiothreitol (DTT).
19. Bovine Serum Albumin (BSA, 20mg/ml).
20. Murine RNase inhibitor (Vazyme biotech).
21. Triton X-100 (molecular biology grade).
22. 10mM dNTPs.
23. 10mM ATPs.
24. Sodium dodecyl sulfate (SDS).
25. Proteinase K.
26. Phenylmethyl sulfonyl fluoride (PMSF).
27. N'N-Dimethylformamide (DMF).
28. Digitonin.
29. Spermidine.
30. T4 Polynucleotide Kinase (NEB).
31. Reverse transcriptase (Thermo recommended).
32. TruePrep DNA Library Prep Kit V2 (Vazyme biotech, cat#TD511, for CH-RNA-seq tagmentation).
33. Transposase Tn5 without adaptor embedding (Vazyme biotech, cat#S111, for CH-ATAC-seq).
34. RNA second strand synthesis module (NEB, cat#E6111L, for CH-RNA-seq).
35. VAHTS DNA Clean Beads (Vazyme Biotech).
36. Ampligase DNA ligase (Lucigen, for CH-ATAC-seq).
37. T4 DNA polymerse (NEB, for CH-ATAC-seq).
38. KAPA HiFi HotStart ReadyMix (Kapa Biosystems).
39. VAHTS® Circularization Kit for MGI (Vazyme).
40. Qubit dsDNA kit (Invitrogen).
41. Qubit ssDNA kit (Invitrogen).
42. Qubit tubes (Invitrogen).
43. DNBSEQ-T7RS high-throughput sequencing kit (MGI, cat#1000016105).


1. Centrifuge.
2. Ice markers.
3. DNase/RNase free Lo-bind 96-well plates.
4. DNase/RNase free Lo-bind centrifuge tubes (1.5ml, 15ml, 50ml).
5. 96-well PCR machine.
6. Incubator.
7. Rotator.
8. Microscope.
9. Multi-channel pipets (10ul, 50ul).
10. Hemocytometer.
11. Self-adhesive Sealing Film for Microplate.
12. Refrigerator (4°C) and freezer (-20°C and -80°C).
13. Qubit 3.0.
14. DNBSEQ-T7 platform (MGI).


Download CH-Protocols

Table 1 oligonucleotide sequences used in CH-seq

Oligonucleotie name Sequence
Download List of all oligonucleotide sequences used in CH-seq

About ACA

We used single-cell RNA sequencing to determine the cell type composition of Axolotl and construct a basic scheme for the Axolotl cell Atlas (ACA). To allow for public access of the resource, we created a Axolotl cell Atlas (ACA) website at http://bis.zju.edu.cn/ACA/.

ACA website consists of seven web pages.
  1. Home: This page contains three sections. About describes the functions and update of the ACA website, CH-seq protocol describes how CH-seq operates.

  2. Atlas:WT : We analysed > 700,000 single cells from 19 neotenic tissues. Tissue datasets are grouped into 70 major clusters and 471 sub-clusters.
META: We analysed ~140,000 single cells from 16 metamorphosed axolotl tissues. Tissue datasets are grouped into 57 major clusters and 304 sub-clusters.
Development: We analysed >210,000 single cells of whole larva axolotl organism span from day30 to day70 postfertilization and generated 35 major clusters.
We created this page to achieve better visualization for global view. You can view each group, each tissue and each gene by choosing the optional box on the left. Atlas view provides global view on single cell level, Marker list provides marker genes for each cluster.

  3. Gallery: In the Gallery, you can download the single-cell digital gene expression (DGE) matrix and get information of the cell number and sample source for each data. The results of the clustering analysis with marker genes are also shared on the Gallery.

  4. Search: You can search genes in the sample you selected, the bar chart of the gene will be returned.

  5. Run-scACA: You can upload the DGE matrix of RNA-seq data (scRNA-seq data or bulk RNA-seq data) and the scACA can help you to identify cell types in your data. The results are showed by interactive heatmap and table in csv format. In heatmap, the row means the defined cell type, the column means the query sample, and the color of block indicates the strength of the correlation. In the result table, Pearson correlation coefficients between query samples (to be identified) and reference cell types are provided.

  6. FAQ: Some frequently asked questions are listed on this page.

  7. Contact: Download links for data and contact information for data providers are placed in this page. We also provide message board on this page.


Fang Ye, Guodong Zhang, Weigao E., Haide Chen, Chengxuan Yu, Lei Yang, Yuting Fu, Jiaqi Li, Sulei Fu, Zhongyi Sun, Lijiang Fei, Qile Guo, Jingjing Wang, Yanyu Xiao, Xinru Wang, Peijing Zhang, Lifeng Ma, Dapeng Ge, Suhong Xu, Juan Caballero-Pérez, Alfredo Cruz-Ramírez, Yincong Zhou, Ming Chen, Ji-Feng Fei, Xiaoping Han and Guoji Guo. Construction of the axolotl cell landscape using combinatorial hybridization sequencing at single-cell resolution. Nature communications, 2022. DOI: 10.1038/s41467-022-31879-z.


On July 23, 2021: ACA Version 0.1 released.