Total RNA was extracted using an RNeasy Mini Kit (Qiagen). For ribosomal RNA depleted total RNA-seq, depletion of rRNA from 1 µg of ES total RNA was performed by using GeneRead rRNA Depletion Kits (Qiagen). For single-cell RamDA-seq, single cells were directly sorted into the cell lysis buffer containing RNasein plus (Promega), Cell Lysis Buffer (Roche), NP40 (Thermo), and RNase free water (TaKaRa). For averaged G1-phase cell lysate sample, 180 cells were sorted into 180 µl of the cell lysis buffer and individually divided into 1 μl in a 96-well PCR plate after well-mixing to lyse the cells. The cell lysate solution was stored at -80ºC until use. For bulk RNA-seq, the libraries were constructed by using a commercial kit (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; NEB) in accordance with the manufacturer’s protocol, with slight modifications during the reverse-transcription and PCR steps. SMART-Seq v4 libraries were prepared according to manufacture's instructions accompanying the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech). For library construction of RamDA-seq and C1-RamDA-seq, refer to the protocol in "Full-length total RNA sequencing from single cells uncovers dynamics of non-polyadenylated RNAs, recursive splicing and enhancer RNAs".
GEO Accession
GSM2608734