My publication on Treg

Immunology

Morikawa, H. et al. Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell-specific transcriptional regulation. Proceedings of the National Academy of Sciences of the United States of America 111, 5289-5294, doi:10.1073/pnas.1312717110 (2014).
: My recent paper on Regulatory T-cells. This paper show the independent roles of Foxp3 and epigenetic modification of the genome (DNA Methylation). Foxp3 is thought to be the master gene of Regulatory T-cells, but from this paper, Foxp3 is essential for gene regulation of regulatory T-cell, but not sufficient.

Morikawa, H. & Sakaguchi, S. Genetic and epigenetic basis of Treg cell development and function: from a FoxP3-centered view to an epigenome-defined view of natural Treg cells. Immunological reviews 259, 192-205, doi:10.1111/imr.12174 (2014).
: Review article on regulatory T-cell specific gene regulation. Main aim of this review is show the latest general understanding of regulatory T-cell specific gene regulation. Understanding of this is important especially for clinical application of human regulatory T-cell, because human regulatory T-cell easily express Foxp3, but they are not all regulatory T-cells.

Textbooks on Bioinfomatics

Introduction of textbooks on bioinfomatics


Well known text book for learning pattern recognition. Easy to read.

Well known text book for statistical learning.

Now I am moving to new server.

As the server response is too slow, I will move to new server.

It takes about 5 days.

Thank you for your patients.

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Moving to new server has completed. Thank you.

Available data of Single cell sequencing

Several papers on single cell sequencing have been published these several years.
You can try to analyze single cell sequence data without getting data by your self.
I here show several available data published in major journals.

Please let me know if you know more data than these listed below.

Let’s enjoy analyzing single cell sequence data!!

1. Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells.

Organism: Mus Musculus
Cell type: BMDCs with LPS
Sample number: 24
Library preparation: Smarter+not Nextera
DATA: SRP015959

Nature. 2013 Jun 13;498(7453):236-40. doi: 10.1038/nature12172. Epub 2013 May 19.

2. Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types.

Organism: Mus Musculus
Cell type: Spleen with LPS
Sample number: 4000
Library preparation: Ido
DATA:SRP035326

Science. 2014 Feb 14;343(6172):776-9. doi: 10.1126/science.1247651.

3. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells.

Organism: Homo sapiens
Cell type: Myoblasts
Sample number: 384
Library preparation: smarter
DATA: SRP033135

Nat Biotechnol. 2014 Apr;32(4):381-6. doi: 10.1038/nbt.2859. Epub 2014 Mar 23.

4. Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq.

Organism: Mus Musculus
Cell type: AT1,AT2,BP in lung
Sample number: 201
Library preparation: Smarter
DATA: SRP033209

Nature. 2014 May 15;509(7500):371-5. doi: 10.1038/nature13173. Epub 2014 Apr 13.

5. Single-cell RNA sequencing reveals T helper cells synthesizing steroids de novo to contribute to immune homeostasis.

Organism: Mus Musculus
Cell type: naïve CD4 T with stm
Sample number: 96
Library preparation: Smarter
DATA: ERP005640

Cell Rep. 2014 May 22;7(4):1130-42. doi: 10.1016/j.celrep.2014.04.011. Epub 2014 May 10.

6. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation.

Organism: Mus Musculus
Cell type: BMDCs with stim
Sample number: 2425
Library preparation: Smarter
DATA: SRP027537

Nature. 2014 Jun 19;510(7505):363-9. doi: 10.1038/nature13437. Epub 2014 Jun 11.

7. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma.

Organism: Homo sapiens
Cell type: Glioblastoma
Sample number: 875
Library preparation: Smarter
DATA: SRP042161

Science. 2014 Jun 20;344(6190):1396-401. doi: 10.1126/science.1254257. Epub 2014 Jun 12.

8. Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing.

Organism: Mus Musculus
Cell type: lumbar dorsal root ganglion
Sample number: 864
Library preparation: Nature Protocols. 2012 . 10.1038/nprot.2012.022
DATA: SRP044898

Nat Neurosci. 2015 Jan;18(1):145-53. doi: 10.1038/nn.3881. Epub 2014 Nov 24.

Single-cell RNA-sequencing technology

Summary of single cell RNA-sequencing technology. Please let me know if there is another methods or if you find some misunderstanding.

a) Smart-seq2 & Smart-seq (= the SMARTer Ultra Low RNA Kit from Clontech)

By reverse-transcription with oligo-dT anchored primer with reverse-transcriptase from the Moloney murine leukemia virus (M-MLV RT), a few non-templated cytosines are added. After second strand synthesis using oligo-dG primer, amplification of dsDNA is performed by PCR. Smart-seq2 is optimized method of Smart-seq, so the basic technology used for library construction is same as previous version. Sensitivity, accuracy and full-length coverage of transcripts are improved.

1. Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nature protocols 9, 171-181, doi:10.1038/nprot.2014.006 (2014). (published from Karolinska Institutet)
2. Ramskold, D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nature biotechnology 30, 777-782, doi:10.1038/nbt.2282 (2012). (published from Karolinska Institutet)

b) Quartz-seq

After reverse-transcription with oligo-dT anchored primer, poly-A is added to the 3′ end of the first strand by terminal deoxynucleotidyl transferase. After second strand synthesis using oligo-dT primer, amplification of dsDNA is performed. The feature of this method is introduction of suppressed PCR (2), which reduce by-product of PCR. This method is published from RIKEN, Japan.

1. Sasagawa, Y. et al. Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogeneity. Genome Biol 14, R31, doi:10.1186/gb-2013-14-4-r31 (2013).
2. Siebert, P. D., Chenchik, A., Kellogg, D. E., Lukyanov, K. A. & Lukyanov, S. A. An improved PCR method for walking in uncloned genomic DNA. Nucleic acids research 23, 1087-1088 (1995).

c) Cel-seq

After reverse-transcription with primer including T7 promoter and oligo-dT, poly-A is added to the 3′ end of the first strand by terminal deoxynucleotidyl transferase. After second strand synthesis using oligo-dT primer, amplification of dsDNA is performed with in vitro transcription by T7 polymerase (IVT). Cel-Seq can produce strand specific information. But the problem is the highly 3′-end skew bias of amplification.

Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-Seq: single-cell RNA-Seq by multiplexed linear amplification. Cell reports 2, 666-673, doi:10.1016/j.celrep.2012.08.003 (2012).

d) Tang’s single cell RNA-seq

After reverse-transcription with oligo-dT anchored primer, poly-A is added to the 3′ end of the first strand by terminal deoxynucleotidyl transferase. After second strand synthesis using oligo-dT primer, amplification of dsDNA is performed by PCR.

Tang, F. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nature methods 6, 377-382, doi:10.1038/nmeth.1315 (2009).

e) STRT

By reverse-transcription with oligo-dT anchored primer with reverse-transcriptase from the Moloney murine leukemia virus (M-MLV RT). As An oligo consisting of guanine residues and barcode are included in the reaction of RT, barcode sequence is added to 5′ end. After performing the biotinated single primer PCR, 5′ end of cDNAs are collected with Streptavidin beads leading to library construction.

Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res 21, 1160-1167, doi:10.1101/gr.110882.110 (2011).

f) Digital quantitative single-cell RNAs-seq

After reverse-transcription with oligo-dT anchored primer, assign unique ID to each product during or after synthesizing dsDNA. By this unique ID tagging, you can know the real number of reads even after PCR amplification.

1. Islam, S. et al. Quantitative single-cell RNA-seq with unique molecular identifiers. Nature methods 11, 163-166, doi:10.1038/nmeth.2772 (2014).
2. Shiroguchi, K., Jia, T. Z., Sims, P. A. & Xie, X. S. Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes. Proceedings of the National Academy of Sciences of the United States of America 109, 1347-1352, doi:10.1073/pnas.1118018109 (2012).
3. Kivioja, T. et al. Counting absolute numbers of molecules using unique molecular identifiers. Nature methods 9, 72-74, doi:10.1038/nmeth.1778 (2012).
4. Casbon, J. A., Osborne, R. J., Brenner, S. & Lichtenstein, C. P. A method for counting PCR template molecules with application to next-generation sequencing. Nucleic acids research 39, e81, doi:10.1093/nar/gkr217 (2011).

Coming New Sequencers

LINKS for coming new sequencers (15th January 2015)
Please let me know if you know another coming sequencer.

2nd generation

GnuBIO
QIAGEN-Intelligent Bio-Systems

3rd generation

NABsys (beta version is available)
Oxford Nanopore Technologies (beta version is available)
GenapSys (beta version is available)
Genia (beta version is available)

Base4 Innovations (nearly practical)
Stratos Genomics (medium stage)

Eve Biomedical (early stage)
Quantapore (early stage)
Quantum Biosystems (early stage)