Details

Autor(en) / Beteiligte

• Boskovic, Nina
• Yazgeldi, Gamze
• Ezer, Sini
• Tervaniemi, Mari H.
• Inzunza, Jose
• Deligiannis, Spyridon Panagiotis
• Yaşar, Barış
• Skoog, Tiina
• Krjutškov, Kaarel
• Katayama, Shintaro
• Kere, Juha

Titel

Optimized single-cell RNA sequencing protocol to study early genome activation in mammalian preimplantation development

Ist Teil von

• STAR protocols, 2023-09, Vol.4 (3), p.102357-102357, Article 102357

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2023

Quelle

Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals

Beschreibungen/Notizen

• Here, we present a modification of single-cell tagged reverse transcription protocol to study gene expression on a single-cell level or with limited RNA input. We describe different enzymes for reverse transcription and cDNA amplification, modified lysis buffer, and additional clean-up steps before cDNA amplification. We also detail an optimized single-cell RNA sequencing method for handpicked single cells, or tens to hundreds of cells, as input material to study mammalian preimplantation development. For complete details on the use and execution of this protocol, please refer to Ezer et al.1 [Display omitted] •Multiplexed RNA-seq library protocol for single embryos or cells•5′ end targeted sequencing for transcription start site capture•Read normalization by spike-in RNAs for accurate quantification of expression•Degrading RNAs distinguished by 3′ predominance of read start sites Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a modification of single-cell tagged reverse transcription protocol to study gene expression on a single-cell level or with limited RNA input. We describe different enzymes for reverse transcription and cDNA amplification, modified lysis buffer, and additional clean-up steps before cDNA amplification. We also detail an optimized single-cell RNA sequencing method for handpicked single cells, or tens to hundreds of cells, as input material to study mammalian preimplantation development.