Small-seq for single-cell small-RNA sequencing
Small RNAs participate in several cellular processes, including splicing, RNA modification, mRNA degradation, and translational arrest. Traditional methods for sequencing small RNAs require a large amount of cell material, limiting the possibilities for single-cell analyses. We describe Small-seq, a...
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| Veröffentlicht in: | Nature protocols 2018-10, Vol.13 (10), p.2407-2424 |
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| creator | Hagemann-Jensen, Michael Abdullayev, Ilgar Sandberg, Rickard Faridani, Omid R |
| description | Small RNAs participate in several cellular processes, including splicing, RNA modification, mRNA degradation, and translational arrest. Traditional methods for sequencing small RNAs require a large amount of cell material, limiting the possibilities for single-cell analyses. We describe Small-seq, a ligation-based method that enables the capture, sequencing, and molecular counting of small RNAs from individual mammalian cells. Here, we provide a detailed protocol for this approach that relies on standard reagents and instruments. The standard protocol captures a complex set of small RNAs, including microRNAs (miRNAs), fragments of tRNAs and small nucleolar RNAs (snoRNAs); however, miRNAs can be enriched through the addition of a size-selection step. Ready-to-sequence libraries can be generated in 2–3 d, starting from cell collection, with additional days needed to computationally map the sequence reads and calculate molecular counts.
Faridani and colleagues describe Small-seq, a protocol for generating sequencing libraries of small RNAs from single cells. |
| doi_str_mv | 10.1038/s41596-018-0049-y |
| format | Article |
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Faridani and colleagues describe Small-seq, a protocol for generating sequencing libraries of small RNAs from single cells.</description><identifier>ISSN: 1754-2189</identifier><identifier>ISSN: 1750-2799</identifier><identifier>EISSN: 1750-2799</identifier><identifier>DOI: 10.1038/s41596-018-0049-y</identifier><identifier>PMID: 30250291</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>631/1647/2017 ; 631/1647/514/1949 ; 631/337/384/521 ; Analytical Chemistry ; Biological Techniques ; Biomedical and Life Sciences ; Computational Biology/Bioinformatics ; Counting ; DNA, Complementary - genetics ; Flow Cytometry - methods ; Gene Library ; Gene sequencing ; HEK293 Cells ; Humans ; Life Sciences ; Mammalian cells ; Medicin och hälsovetenskap ; Methods ; Microarrays ; miRNA ; mRNA ; Nucleoli ; Organic Chemistry ; Polymerase Chain Reaction - methods ; Protocol ; Reagents ; Ribonucleic acid ; RNA ; RNA modification ; RNA sequencing ; RNA, Small Untranslated - genetics ; RNA, Small Untranslated - isolation & purification ; Sequence Analysis, RNA - methods ; Single-Cell Analysis - methods ; Splicing</subject><ispartof>Nature protocols, 2018-10, Vol.13 (10), p.2407-2424</ispartof><rights>The Author(s), under exclusive licence to Springer Nature Limited 2018</rights><rights>COPYRIGHT 2018 Nature Publishing Group</rights><rights>Copyright Nature Publishing Group Oct 2018</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c494t-70e8d4a0428765d9e5960f2cee24269b684ebbd1e739eda5c0c72c8051e5634d3</citedby><cites>FETCH-LOGICAL-c494t-70e8d4a0428765d9e5960f2cee24269b684ebbd1e739eda5c0c72c8051e5634d3</cites><orcidid>0000-0001-6473-1740 ; 0000-0001-9330-887X ; 0000-0002-6423-8216</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,315,781,785,886,27929,27930</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30250291$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttp://kipublications.ki.se/Default.aspx?queryparsed=id:139292263$$DView record from Swedish Publication Index$$Hfree_for_read</backlink></links><search><creatorcontrib>Hagemann-Jensen, Michael</creatorcontrib><creatorcontrib>Abdullayev, Ilgar</creatorcontrib><creatorcontrib>Sandberg, Rickard</creatorcontrib><creatorcontrib>Faridani, Omid R</creatorcontrib><title>Small-seq for single-cell small-RNA sequencing</title><title>Nature protocols</title><addtitle>Nat Protoc</addtitle><addtitle>Nat Protoc</addtitle><description>Small RNAs participate in several cellular processes, including splicing, RNA modification, mRNA degradation, and translational arrest. Traditional methods for sequencing small RNAs require a large amount of cell material, limiting the possibilities for single-cell analyses. We describe Small-seq, a ligation-based method that enables the capture, sequencing, and molecular counting of small RNAs from individual mammalian cells. Here, we provide a detailed protocol for this approach that relies on standard reagents and instruments. The standard protocol captures a complex set of small RNAs, including microRNAs (miRNAs), fragments of tRNAs and small nucleolar RNAs (snoRNAs); however, miRNAs can be enriched through the addition of a size-selection step. Ready-to-sequence libraries can be generated in 2–3 d, starting from cell collection, with additional days needed to computationally map the sequence reads and calculate molecular counts.
Faridani and colleagues describe Small-seq, a protocol for generating sequencing libraries of small RNAs from single cells.</description><subject>631/1647/2017</subject><subject>631/1647/514/1949</subject><subject>631/337/384/521</subject><subject>Analytical Chemistry</subject><subject>Biological Techniques</subject><subject>Biomedical and Life Sciences</subject><subject>Computational Biology/Bioinformatics</subject><subject>Counting</subject><subject>DNA, Complementary - genetics</subject><subject>Flow Cytometry - methods</subject><subject>Gene Library</subject><subject>Gene sequencing</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>Life Sciences</subject><subject>Mammalian cells</subject><subject>Medicin och hälsovetenskap</subject><subject>Methods</subject><subject>Microarrays</subject><subject>miRNA</subject><subject>mRNA</subject><subject>Nucleoli</subject><subject>Organic Chemistry</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Protocol</subject><subject>Reagents</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA modification</subject><subject>RNA sequencing</subject><subject>RNA, Small Untranslated - 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Academic</collection><collection>SwePub</collection><collection>SwePub Articles</collection><jtitle>Nature protocols</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hagemann-Jensen, Michael</au><au>Abdullayev, Ilgar</au><au>Sandberg, Rickard</au><au>Faridani, Omid R</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Small-seq for single-cell small-RNA sequencing</atitle><jtitle>Nature protocols</jtitle><stitle>Nat Protoc</stitle><addtitle>Nat Protoc</addtitle><date>2018-10-01</date><risdate>2018</risdate><volume>13</volume><issue>10</issue><spage>2407</spage><epage>2424</epage><pages>2407-2424</pages><issn>1754-2189</issn><issn>1750-2799</issn><eissn>1750-2799</eissn><abstract>Small RNAs participate in several cellular processes, including splicing, RNA modification, mRNA degradation, and translational arrest. Traditional methods for sequencing small RNAs require a large amount of cell material, limiting the possibilities for single-cell analyses. We describe Small-seq, a ligation-based method that enables the capture, sequencing, and molecular counting of small RNAs from individual mammalian cells. Here, we provide a detailed protocol for this approach that relies on standard reagents and instruments. The standard protocol captures a complex set of small RNAs, including microRNAs (miRNAs), fragments of tRNAs and small nucleolar RNAs (snoRNAs); however, miRNAs can be enriched through the addition of a size-selection step. Ready-to-sequence libraries can be generated in 2–3 d, starting from cell collection, with additional days needed to computationally map the sequence reads and calculate molecular counts.
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| subjects | 631/1647/2017 631/1647/514/1949 631/337/384/521 Analytical Chemistry Biological Techniques Biomedical and Life Sciences Computational Biology/Bioinformatics Counting DNA, Complementary - genetics Flow Cytometry - methods Gene Library Gene sequencing HEK293 Cells Humans Life Sciences Mammalian cells Medicin och hälsovetenskap Methods Microarrays miRNA mRNA Nucleoli Organic Chemistry Polymerase Chain Reaction - methods Protocol Reagents Ribonucleic acid RNA RNA modification RNA sequencing RNA, Small Untranslated - genetics RNA, Small Untranslated - isolation & purification Sequence Analysis, RNA - methods Single-Cell Analysis - methods Splicing |
| title | Small-seq for single-cell small-RNA sequencing |
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