GSM2908219: C1_RamDA_mESC_10pg_CF04; Mus musculus; RNA-Seq
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 "Single-cell full-length total RNA sequencing uncovers dynamics of non-polyadenylated RNAs, recursive splicing and enhancer RNAs". For rRNA-depleted RNA-seq using 1 and 10 ng of diluted total RNA, sequencing libraries were prepared with KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems) according to the manufacturer's protocol. C1-SMART-Seq v4 libraries were prepared using Fluidigm C1 with the script "Full-length mRNA Sequencing" was downloaded from Script Hub (https://jp.fluidigm.com/c1openapp/scripthub)in accordance with the manufacturer's protocol. For analyses of plate-to-plate variability (batch effect) of RamDA-seq, we prepared RamDA-seq library DNA by using the advanced method of RamDA-seq (refer to the protocol in "Single-cell full-length total RNA sequencing uncovers dynamics of non-polyadenylated RNAs, recursive splicing and enhancer RNAs").
GEO Accession
GSM2908219