GSM6820307: mES cells Dis3L KO_3; Mus musculus; ssRNA-seq
RNA from ES cells was extracted by lysis of 1 million cells using TRI Reagent, following manufacturers protocol. RNA from blastocysts was extracted using PicoPure RNA Isolation Kit following manufacturers protocol. For ES cells' RNA sequencing, total DNA-free RNA was ribodepleted using Ribo-Zero Gold rRNA-removal kit (Illumina). rRNA-free RNA samples were clean-up by precipitation with 3M NaAc. Sequencing libraries were prepared using KAPA Stranded RNA-Seq Library Preparation Kit (KAPA Biosystems). Between each step samples were cleaned up using AMPure XP Reagent magnetics beads. Library quality and fragment size distribution was verified by electrophoresis in Agilent 2100 Bioanalyzer (Agilent Technologies Inc.).     RNA-seq libraries from single blastocysts DNA-free RNA samples were prepared by tagmentation reaction following published protocols (24,25) with various steps and amount of enzyme optimized for use of homemade batch of Tn5 transposase produced in our laboratory. Briefly: Tn5 (0,25 mg/ml) was loaded with linker oliguncleotides Tn5ME-A/Tn5Merev and Tn5ME-B/Tn5Merev by mixing of 10 ul Tn5 with 0,5 ul of both linkers (0,35 uM) and incubating for 45 min in 23oC with shaking at 350 rpm. Right before the reaction setup loaded Tn5 was diluted 10 times with nuclease-free water. 10 ul of freshly prepared tagmentation buffer (20 mM Tris-HCl ph 7,5; 20 mM MgCl2; 50% dimethylformamide (Sigma-Aldritch)  were mixed with 5 ul of diluted Tn5 and 5 ul od cDNA, incubated for 3 min at 55oC in preheated thermocycler and cooled to 10oC for 1 min. Reaction was inactivated by addition of 5 ul 0,2% SDS (Sigma-Aldrich and incubation for 5 min at RT. Tagmented cDNA was purified in 1,25 volume of AMPure XP Reagent magnetic beads.     For library amplification (15 cycles) KAPA HiFi HotStart ReadyMix PCR Kit (Roche) with addition of 5% DMSO was used. Number of cycles yielding best results was determined experimentally and was further individually adjusted for each batch of prepared cDNA. Library quality and fragment size distribution was verified by electrophoresis in Agilent 2100 Bioanalyzer.  Illumina NovaSeq or NextSeq sequencing platform in the 100-nt or 75-nt pair-end mode was used for sequencing.