title: GSM3666472: MeCP2_KO_PROseq_rep2; Mus musculus; OTHER
library_construction_protocol: Forebrain (cortex and hippocampus) was dissected from 8-to-12-week-old MeCP2 KO, MeCP2 R306C, Dnmt3a cKO mice and their respective WT littermate controls. Tissue was homogenized in ice-cold PBS using a Dounce homogenizer, and nuclei were pelleted by spinning at 17,000 g for 5 minutes at 4°C. Chromatin isolation was performed as previously described (Chu et al., 2018). Nuclei were resuspended in lysis buffer (NUN: 0.3 M NaCl, 1 M Urea, 20 mM HEPES pH 7.5, 7.5 mM MgCl2, 0.2 mM EDTA, 1% NP-40, 1 mM DTT, cOmplete Protease Inhibitor (Roche), 40 units/mL SUPERase In RNase Inhibitor (Invitrogen)) and incubated on ice for 30 minutes. Lysed nuclei were centrifuged at 12,500 g for 30 minutes at 4°C. The resulting insoluble chromatin pellet was washed in 50 mM Tris-HCl pH 7.5 and placed in 50 μL chromatin storage buffer (25% glycerol, 50 mM Tris-HCl pH 8.0, 5 mM MgAc2, 0.1 mM EDTA, 5 mM DTT, 40 units/mL SUPERase In RNase Inhibitor). Chromatin was solubilized by sonicating on a Diagenode Bioruptor (High Power, 40 cycles, 30 seconds on/30 seconds off). Solubilized chromatin was flash-frozen in liquid nitrogen and stored at -80 °C. PRO-seq RNA purification was performed as previously described (Kwak et al., 2013; Mahat et al., 2016). Frozen chromatin was thawed on ice and combined with 50 μL 2X Run-on Buffer (10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 300 mM KCl, 40 μM Biotin-11-ATP, 40 μM Biotin-11-GTP, 40 μM Biotin-11-CTP, 40 μM Biotin-11-UTP, 1% Sarksosyl, 1 mM DTT, 0.8 units/μL SUPERase In RNase Inhibitor). The reaction was incubated at 37°C for 5 minutes and then quenched with 500 μL Trizol LS, combined with 130 μL chloroform, and centrifuged at 17,000g for 5 minutes at 4 °C. The aqueous layer containing RNA was combined with 1 mL ice-cold ethanol and 2.5 μL GlycoBlue (Invitrogen) and centrifuged at 17,000g for 15 minutes at 4 °C. The RNA pellet was washed in 75% ethanol, air-dried, and resuspended in 20 μL nuclease-free water. RNA was hydrolyzed by adding 5 μL 1 N NaOH and incubating for 4 minutes at 4°C, then neutralized with 30 μL 1 M Tris-HCl pH 7.0. Hydrolyzed RNA was run on a Micro Bio-Spin P-30 Column (Bio-Rad) to remove excess salts. Biotinylated RNA was purified with magnetic streptavidin beads (NEB) at room temperature for 20 minutes and washed 2 times each with 500 μL High-Salt (2 M NaCl, 50 mM Tris-HCl pH 7.4, 0.5% Triton X-100, 4 units/mL SUPERase In RNase Inhibitor), Binding (300 mM NaCl, 10 mM Tris-HCl pH 7.4, 0.1% Triton X-100, 4 units/mL SUPERase In RNase Inhibitor), and Low-Salt (5 mM Tris-HCl pH 7.4, 0.1% Triton X-100, 4 units/mL SUPERase In RNase Inhibitor) wash buffers. Beads were resuspended in 300 μL Trizol, and combined with 60 μL chloroform, and RNA was purified as above. PRO-seq library preparation was performed as previously described (Kwak et al., 2013; Mahat et al., 2016). The dried RNA pellet was resuspended in 4 μL of 12.5 μM Reverse 3' RNA adaptor and used to make a T4 RNA Ligase I reaction mix (5 μM Reverse 3' RNA Adaptor, 1X T4 RNA Ligase I Buffer, 1 mM ATP, 10 units T4 RNA Ligase I, 10% PEG 8000, 2 units/μL SUPERase In RNase Inhibitor). The reaction was incubated overnight at 20°C. The ligated RNA was purified with magnetic streptavidin beads, washed, and extracted with Trizol as above. The RNA was treated with RNA 5' Pyrophosphohydrolase (RppH) to remove the 5' cap (1X NEBuffer 2, 10 units RppH, 0.2 units/μL SUPERase In RNase Inhibitor) and incubated for 1 hour at 37°C. A hydroxyl repair reaction mix (1X PNK Buffer, 1 mM ATP, 25 units PNK, 0.2 units/μL SUPERase In RNase Inhibitor) was added directly to the Rpph reaction and incubated for an additional hour at 37°C. RNA was extracted with Trizol as above. The dried RNA pellet was resuspended in 4 μL of 12.5 μM Reverse 5' RNA adaptor and used to make a T4 RNA Ligase I reaction mix (5 μM Reverse 5' RNA Adaptor, 1X T4 RNA Ligase I Buffer, 1 mM ATP, 10 units T4 RNA Ligase I, 10% PEG 8000, 2 units/μL SUPERase In RNase Inhibitor). The reaction was incubated overnight at 20°C. The ligated RNA was purified with magnetic streptavidin beads, washed, and extracted with Trizol as above. The resulting RNA library was reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen). The cDNA libraries were amplified using Q5 DNA Polymerase (NEB) for 14 cycles or more, as determined by a test amplification with diluted cDNA. Amplified libraries were purified on Agencourt RNAClean XP beads. PRO-seq
attributes.tag: GEO Accession
attributes.value: GSM3666472