54. Guo H, Zhu P, Guo F, Li X, Wu X, Fan X, Wen L, Tang F (2015) Profiling DNA methylome
landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing.
Nat Protoc 10(5):645–659. https://doi.org/10.1038/nprot.2015.039
55. Smallwood SA, Lee HJ, Angermueller C, Krueger F, Saadeh H, Peat J, Andrews SR, Stegle O,
Reik W, Kelsey G (2014) Single-cell genome-wide bisulfite sequencing for assessing epigenetic
heterogeneity. Nat Methods 11(8):817–820. https://doi.org/10.1038/nmeth.3035
56. Clark SJ, Smallwood SA, Lee HJ, Krueger F, Reik W, Kelsey G (2017) Genome-wide baseresolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing
(scBS-seq). Nat Protoc 12(3):534–547. https://doi.org/10.1038/nprot.2016.187
57. Luo C, Keown CL, Kurihara L, Zhou J, He Y, Li J, Castanon R, Lucero J, Nery JR, Sandoval
JP, Bui B, Sejnowski TJ, Harkins TT, Mukamel EA, Behrens MM, Ecker JR (2017) Single-cell
methylomes identify neuronal subtypes and regulatory elements in mammalian cortex. Science
357(6351):600–604. https://doi.org/10.1126/science.aan3351
58. Angermueller C, Clark SJ, Lee HJ, Macaulay IC, Teng MJ, Hu TX, Krueger F, Smallwood S,
Ponting CP, Voet T, Kelsey G, Stegle O, Reik W (2016) Parallel single-cell sequencing links
transcriptional and epigenetic heterogeneity. Nat Methods 13(3):229–232. https://doi.org/10.
1038/nmeth.3728
59. Han J, Zhang Z, Wang K (2018) 3C and 3C-based techniques: the powerful tools for spatial
genome organization deciphering. Mol Cytogenet 11:21. https://doi.org/10.1186/s13039-0180368-2
60. Li G, Cai L, Chang H, Hong P, Zhou Q, Kulakova EV, Kolchanov NA, Ruan Y (2014)
Chromatin interaction analysis with paired-end tag (ChIA-PET) sequencing technology and
application. BMC Genomics 15(Suppl 12):S11. https://doi.org/10.1186/1471-2164-15-S12S11
61. Mumbach MR, Rubin AJ, Flynn RA, Dai C, Khavari PA, Greenleaf WJ, Chang HY (2016)
HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nat Methods
13(11):919–922. https://doi.org/10.1038/nmeth.3999
62. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL,
Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification
of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179. https://doi.org/10.1093/
nar/gni178
63. Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP (2007) Protocol: a highly sensitive
RT-PCR method for detection and quantification of microRNAs. Plant Methods 3:12. https://
doi.org/10.1186/1746-4811-3-12
64. Jacobsen N, Andreasen D, Mouritzen P (2011) Profiling microRNAs by real-time PCR.
Methods Mol Biol 732:39–54. https://doi.org/10.1007/978-1-61779-083-6_4
65. Campomenosi P, Gini E, Noonan DM, Poli A, D'Antona P, Rotolo N, Dominioni L, Imperatori
A (2016) A comparison between quantitative PCR and droplet digital PCR technologies for
circulating microRNA quantification in human lung cancer. BMC Biotechnol 16(1):60. https://
doi.org/10.1186/s12896-016-0292-7
66. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ,
Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW,
Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley
TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP,
Romine S, Milanovich FP, White HE, Regan JF, Karlin-Neumann GA, Hindson CM,
Saxonov S, Colston BW (2011) High-throughput droplet digital PCR system for absolute
quantitation of DNA copy number. Anal Chem 83(22):8604–8610. https://doi.org/10.1021/
ac202028g
67. Song Y, Kilburn D, Song JH, Cheng Y, Saeui CT, Cheung DG, Croce CM, Yarema KJ, Meltzer
SJ, Liu KJ, Wang TH (2017) Determination of absolute expression profiles using multiplexed
miRNA analysis. PLoS One 12(7):e0180988. https://doi.org/10.1371/journal.pone.0180988
558
C. Dell’Aversana et al.
landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing.
Nat Protoc 10(5):645–659. https://doi.org/10.1038/nprot.2015.039
55. Smallwood SA, Lee HJ, Angermueller C, Krueger F, Saadeh H, Peat J, Andrews SR, Stegle O,
Reik W, Kelsey G (2014) Single-cell genome-wide bisulfite sequencing for assessing epigenetic
heterogeneity. Nat Methods 11(8):817–820. https://doi.org/10.1038/nmeth.3035
56. Clark SJ, Smallwood SA, Lee HJ, Krueger F, Reik W, Kelsey G (2017) Genome-wide baseresolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing
(scBS-seq). Nat Protoc 12(3):534–547. https://doi.org/10.1038/nprot.2016.187
57. Luo C, Keown CL, Kurihara L, Zhou J, He Y, Li J, Castanon R, Lucero J, Nery JR, Sandoval
JP, Bui B, Sejnowski TJ, Harkins TT, Mukamel EA, Behrens MM, Ecker JR (2017) Single-cell
methylomes identify neuronal subtypes and regulatory elements in mammalian cortex. Science
357(6351):600–604. https://doi.org/10.1126/science.aan3351
58. Angermueller C, Clark SJ, Lee HJ, Macaulay IC, Teng MJ, Hu TX, Krueger F, Smallwood S,
Ponting CP, Voet T, Kelsey G, Stegle O, Reik W (2016) Parallel single-cell sequencing links
transcriptional and epigenetic heterogeneity. Nat Methods 13(3):229–232. https://doi.org/10.
1038/nmeth.3728
59. Han J, Zhang Z, Wang K (2018) 3C and 3C-based techniques: the powerful tools for spatial
genome organization deciphering. Mol Cytogenet 11:21. https://doi.org/10.1186/s13039-0180368-2
60. Li G, Cai L, Chang H, Hong P, Zhou Q, Kulakova EV, Kolchanov NA, Ruan Y (2014)
Chromatin interaction analysis with paired-end tag (ChIA-PET) sequencing technology and
application. BMC Genomics 15(Suppl 12):S11. https://doi.org/10.1186/1471-2164-15-S12S11
61. Mumbach MR, Rubin AJ, Flynn RA, Dai C, Khavari PA, Greenleaf WJ, Chang HY (2016)
HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nat Methods
13(11):919–922. https://doi.org/10.1038/nmeth.3999
62. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL,
Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification
of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179. https://doi.org/10.1093/
nar/gni178
63. Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP (2007) Protocol: a highly sensitive
RT-PCR method for detection and quantification of microRNAs. Plant Methods 3:12. https://
doi.org/10.1186/1746-4811-3-12
64. Jacobsen N, Andreasen D, Mouritzen P (2011) Profiling microRNAs by real-time PCR.
Methods Mol Biol 732:39–54. https://doi.org/10.1007/978-1-61779-083-6_4
65. Campomenosi P, Gini E, Noonan DM, Poli A, D'Antona P, Rotolo N, Dominioni L, Imperatori
A (2016) A comparison between quantitative PCR and droplet digital PCR technologies for
circulating microRNA quantification in human lung cancer. BMC Biotechnol 16(1):60. https://
doi.org/10.1186/s12896-016-0292-7
66. Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ,
Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW,
Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley
TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP,
Romine S, Milanovich FP, White HE, Regan JF, Karlin-Neumann GA, Hindson CM,
Saxonov S, Colston BW (2011) High-throughput droplet digital PCR system for absolute
quantitation of DNA copy number. Anal Chem 83(22):8604–8610. https://doi.org/10.1021/
ac202028g
67. Song Y, Kilburn D, Song JH, Cheng Y, Saeui CT, Cheung DG, Croce CM, Yarema KJ, Meltzer
SJ, Liu KJ, Wang TH (2017) Determination of absolute expression profiles using multiplexed
miRNA analysis. PLoS One 12(7):e0180988. https://doi.org/10.1371/journal.pone.0180988
558
C. Dell’Aversana et al.