title: GSM4680413: WT_16-med-am-ATAC; Mus musculus; ATAC-seq
library_construction_protocol: DNA and RNA were isolated using TRIzol reagent (Sigma Aldrich, Darmstadt, Germany) and further AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) purification, following the manufacturer's instructions. For RNA isolation (RNase-free DNAse set, Qiagen) DNAse I digestion was performed on the column. Proteinase K digestion was applied for RNA and DNA isolation (Puregene Proteinase K, Qiagen). Prior to library preparation, DNA and RNA quality was checked by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified by Qubit fluorimetry (Thermo Scientific, Wilmington, USA). All RNA samples reached an RNA integrity number (RIN) > 8.5. ATAC libraries have been prepared according to the Omni-ATAC protocol (Corces et al., 2017a) with minor modifications. In short, 50000 viable cells were pelleted and wahsed in PBS. Subsequently, nuclei were isolated using cold lysis buffer (0.01% Digitonin [Sigma-Aldrich, Munich, Germany], 1% NP40 [Genaxxon Bioscience, Ulm, Germany], 0.1% Tween-20 [Sigma-Aldrich, Munich, Germany]). Resuspension of the nuclei was done in ATAC washing buffer (10 mM Tris-HCl pH 7.4 [Sigma-Aldrich, Munich, Germany], 10 mM NaCl [Sigma-Aldrich, Munich, Germany], 3 mM MgCl2 [Sigma-Aldrich, Munich, Germany]).The tagmentation reaction was performed in 2x transposition buffer (20 mM Tris-HCl pH 7.6 [Sigma-Aldrich, Munich, Germany], 3 mM MgCl2 [Sigma-Aldrich, Munich, Germany], 20% Dimethyl Formamide [Sigma-Aldrich, Munich, Germany]) by the addition of 2.5µl of Tagment DNA Enzyme 1 (Illumina, San Diego, CA) and rotating the mixture at 1000 rpm for 30 minutes at 37°C. The reaction was stopped by the addition of 20µl of 5M Guanidinium thiocyanate (Sigma-Aldrich, Munich, Germany). Transposed chromatin was then purified using 30µl of AMPure XP beads beads (Beckman Coulter, Brea, CA)), and 110 µl of PEG buffer (2.5 M NaCl [Sigma-Aldrich, Munich, Germany], 20% PEG 8000 [Sigma-Aldrich, Munich, Germany]). Library amplification was separated in a two-step procedure. First, 25 µl of NEBNext High Fidelity 2x Master Mix (NEB, Ipswich, MA), 0.8 µl of 10 µM Custom Nextera PCR Primer 1, and 0.8 µl of 10 µM Custom Nextera PCR Barcode was added to 25µl of the transposed DNA, using the following program: 5 minutes at 72°C, 30 seconds at 98°C, 5 cycles of 10 seconds at 98°C, 30 seconds at 63°C, and 1 minute at 72°C, and, finally, 1 minute at 72°C. 5µl of the pre-amplified PCR mixture were used to determine how many additional cycles were needed to reach sufficient amplification of each library. For this, Sybr Green I nucleic acid gel stain (Thermo Fisher Scientific, Waltham, MA) was added and quantitative PCR performed with light cycler instrument (Roche, Basel, Switzerland) by applying the following program: 30 seconds at 98°C, 20 cycles of 10 seconds at 98°C, 30 seconds at 63°C, 1 minutes at 72°C, and, finally, 1 minute at 72°C. In a second step, the previous identified PCR cycles were applied to the remaining pre-amplified mixture. The libraries were then purified with a left sided size selection applying 1.4x of AMPure XP beads (Beckman Coulter, Brea, CA) and finally resuspended in 1x elution buffer (Qiagen, Hilden, Germany). Fragment size distribution was checked by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and concentrations were quantified by Qubit fluorimetry (Thermo Scientific, Wilmington, USA). Sequencing was performed in multiplexes at the DKFZ Genomics and Proteomics Core Facility using the HiSeq 2000 v4 paired-end 125 bp platform (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4680413