title: GSM3733803: G1sE125_R3_RNArep1 [MS25_KO_R3_12.5d]; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted followed by manufacturer's instructions of TRIzol (Invitrogen). DNA after ATAC protocol was isolated using QIAGEN MiniElute PCR purification kit. DNA after CUT&RUN protocol was isolated using phenol-chloroform-isoamyl alcohol method. To generate RNA sequencing libraries, RNA quality was determined with the Agilent Bioanalyzer 2100, accepting RNA integrity numbers (RIN) of > 7 and quantified using Qubit. Directional mRNA libraries were prepared using Illumina TruSeq mRNA Sample Preparation Kits per manufacturer's instructions. Briefly, polyadenylated mRNAs were captured from total RNA using oligo-dT selection. Next, samples were converted to cDNA by reverse transcription, and each sample was ligated to Illumina sequencing adapters containing unique barcode sequences. Barcoded samples were then amplified by PCR and the resulting cDNA libraries by quantified using qPCR. CUT&RUN-sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina complemented with NEBNext Multiplex Oligos for Illumina. The dA-tailing temperature was decreased to 50oC, and the reaction time was increased to 1 hour to compensate for lower enzymatic activity. Next, 2.1x AMPure XP beads (Beckman) were added for cleanup of adaptor-ligated DNA without size selection. After PCR amplification, the reaction was cleaned up with AMPure XP beads. Libraries were validated using the Agilent High Sensitivity DNA Kit on Bioanalyzer. Cleavage Under Targets and Release Using Nuclease (CUT&RUN)
attributes.tag: GEO Accession
attributes.value: GSM3733803