GSM2899073: totalES_03; Mus musculus; RNA-Seq
Total RNA was extracted using a Direct-zol RNA MiniPrep Kit (Zymo Research). We prepared RT-RamDA sequencing library from single-stranded amplified cDNA by using a DNA SMART ChIP-Seq Kit (Clontech). Before performing DNA SMART ChIP-Seq Kit, for digesting remaining RNA, 1 µl of RNase mix [2.5 x NEB buffer 3 (New England Biolabs), 0.1 U RNase H (Thermo Fisher) and RNase If (New England Biolabs) in RNase-free water] was added to 3 µl of RT-RamDA product from diluted 10 pg of total mESC RNA. RNase treatment was carried out at 37°C for 30 min. After RNase treatment, immediately cDNAs were purified by using 12 µl of AMPure XP SPRI beads (Beckman Coulter). Washed AMPure XP beads attached to single-stranded cDNAs were directly eluted using 8 μl of 1 × DNA Dilution Buffer (Clontech) and mixed well using a vortex mixer and pipetting. Sequencing library preparation was performed according to the manual instructions, except that the reaction volume was modified to 1/2.5. Seventeen cycles of PCR were applied for the library DNA. When we performed ribosomal RNA depleted total RNA-seq using diluted 200 ng of total mESC RNA, we utilized a KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems) for the preparation of sequencing library DNA according to the manual instructions. Seven cycles of PCR were applied for the library DNA. Quality control of sequencing library DNA was carried out using a Bioanalyzer Agilent High-Sensitivity DNA Kit (Agilent) and a KAPA library quantification kit (Kapa Biosystems).
GEO Accession
GSM2899073