title: GSM4273506: NET-seq_Control_rep1; Mus musculus; OTHER
library_construction_protocol: The cell fractionation was performed as described previously (Mayer & Churchman, 2016). 10 million ES cells are washed with 500 ml of pre-cooled 1x PBS, resuspended in 150 ml Cytoplasmic lysis buffer (0.15% (v/v) NP-40, 10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25 mM a-amanitin (Sigma), 50 U SUPERase.In (Life Technologies), x1 Halt Protease Inhibitor cocktail mix (ThermoFisher 87786) and incubated on ice for 5 min. The cell lysate is layered over 400 ml of Sucrose buffer (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (w/v) sucrose, 25 mM a-amanitin, 50 U SUPERase.In, x1 Halt Protease Inhibitor cocktail mix) and centrifuged at 16,000 g for 10 min at 4C. The nuclei pellet is resuspended in 500 ml Nuclei wash buffer (0.1% (v/v) Triton X-100, 1 mM EDTA, in 1x PBS, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix) and centrifuged at 1,150 g for 1 min at 4C. Washed nuclei are resuspended in 200 ml Glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (v/v) glycerol, 0.85 mM DTT, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix). Next, 200 ml of Nuclei lysis buffer (1% (v/v) NP-40, 20 mM HEPES pH 7.5, 300 mM NaCl, 1M Urea, 0.2 mM EDTA, 1 mM DTT, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix) are added, mixed by pulsed vortexing and incubated on ice for 2 min. The lysate is centrifuged at 18,500 g for 2 min at 4C. The chromatin pellet is resuspended in 50 ml Chromatin resuspension solution (25 mM a-amanitin, 50 Units SUPERase.In, 1x Protease inhibitor mix in 1x PBS) before RNA preparation. Biological replicates were obtained from two independent Control and dTAG-13 treated cells. Prior to library preparation, SIRV spike-in controls (1:10,000 dilution) were added to the extracted RNA to account for loss of RNA during library preparation. RNA preparation and NET-seq library construction was conducted as originally described (Mylonas).  More than 90% recovery of ligated RNA and cDNA was achieved from 15% TBE-Urea (Invitrogen) and 10% TBE-Urea (Invitrogen), respectively, by adding RNA recovery buffer (R1070-1-10; Zymo Research) to the excised gel slices and further incubating at 70°C (1,500 rpm) for 15 min. Gel slurry was transferred through a ZymoSpin IV Column (C1007-50; Zymo Research) and further precipitated for subsequent library preparation steps. cDNA containing the 3' end sequences of a subset of mature and heavily sequenced snRNAs, snoRNAs, and rRNAs were specifically depleted using biotinylated DNA oligos (Mylonas). Oligo-depleted circularized cDNA was amplified by PCR and double-stranded DNA was run on a 3% low melt agarose gel. The final NET-seq library running at ~150 bp was extracted and further purified using the ZymoClean Gel DNA recovery kit (Zymo Research). Sample purity and concentration was assessed in a 2200 TapeStation and further deep sequenced in a HiSeq 2500 Illumina Platform  The cell fractionation was performed as described previously (Mayer & Churchman, 2016). 10 million ES cells are washed with 500 ml of pre-cooled 1x PBS, resuspended in 150 ml Cytoplasmic lysis buffer (0.15% (v/v) NP-40, 10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25 mM a-amanitin (Sigma), 50 U SUPERase.In (Life Technologies), x1 Halt Protease Inhibitor cocktail mix (ThermoFisher 87786) and incubated on ice for 5 min. The cell lysate is layered over 400 ml of Sucrose buffer (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (w/v) sucrose, 25 mM a-amanitin, 50 U SUPERase.In, x1 Halt Protease Inhibitor cocktail mix) and centrifuged at 16,000 g for 10 min at 4C. The nuclei pellet is resuspended in 500 ml Nuclei wash buffer (0.1% (v/v) Triton X-100, 1 mM EDTA, in 1x PBS, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix) and centrifuged at 1,150 g for 1 min at 4C. Washed nuclei are resuspended in 200 ml Glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (v/v) glycerol, 0.85 mM DTT, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix). Next, 200 ml of Nuclei lysis buffer (1% (v/v) NP-40, 20 mM HEPES pH 7.5, 300 mM NaCl, 1M Urea, 0.2 mM EDTA, 1 mM DTT, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix) are added, mixed by pulsed vortexing and incubated on ice for 2 min. The lysate is centrifuged at 18,500 g for 2 min at 4C. The chromatin pellet is resuspended in 50 ml Chromatin resuspension solution (25 mM a-amanitin, 50 Units SUPERase.In, 1x Protease inhibitor mix in 1x PBS) before RNA preparation. Biological replicates were obtained from two independent Control and dTAG-13 treated cells. Prior to library preparation, SIRV spike-in controls (1:10,000 dilution) were added to the extracted RNA to account for loss of RNA during library preparation. RNA preparation and NET-seq library construction was conducted as originally described (Mylonas).  More than 90% recovery of ligated RNA and cDNA was achieved from 15% TBE-Urea (Invitrogen) and 10% TBE-Urea (Invitrogen), respectively, by adding RNA recovery buffer (R1070-1-10; Zymo Research) to the excised gel slices and further incubating at 70°C (1,500 rpm) for 15 min. Gel slurry was transferred through a ZymoSpin IV Column (C1007-50; Zymo Research) and further precipitated for subsequent library preparation steps. cDNA containing the 3' end sequences of a subset of mature and heavily sequenced snRNAs, snoRNAs, and rRNAs were specifically depleted using biotinylated DNA oligos (Mylonas). Oligo-depleted circularized cDNA was amplified by PCR and double-stranded DNA was run on a 3% low melt agarose gel. The final NET-seq library running at ~150 bp was extracted and further purified using the ZymoClean Gel DNA recovery kit (Zymo Research). Sample purity and concentration was assessed in a 2200 TapeStation and further deep sequenced in a HiSeq 2500 Illumina Platform
attributes.tag: GEO Accession
attributes.value: GSM4273506