title: GSM3666506: MeCP2_KO_0min_FP_PROseq_rep3; Mus musculus; OTHER
library_construction_protocol: One side of each forebrain was used for PRO-seq. Cells were permeabilized as described previously (Mahat et al., 2016), with minor modifications. Tissue was dounce homogenized in 5 mL cold PBS, then spun at 800g for 4 minutes at 4°C. The pellet was washed with 10 mL PBS, then spun at 800g for 4 minutes at 4°C. The pellet was resuspended in 1 mL Buffer W (10 mM Tris pH 8, 10 mM KCl, 250 mM sucrose, 5 mM MgCl2, 0.5 mM DTT, 10% glycerol) and then 9 mL Buffer P was added (Buffer W with 0.1% Igepal). Lysate was filtered through a 40μm cell strainer and spun at 800g for 4 minutes at 4°C. The pellet was resuspended in 1 mL Buffer W and then 9 mL Buffer P was added. Lysate was incubated on ice for 1 minute, then spun at 800g for 4 minutes at 4°C. The pellet was resuspended in 500 μL Buffer F (50 mM Tris pH 8, 40% glycerol, 5 mM MgCl2, 0.5 mM DTT) and spun at 1000g for 4 minutes at 4°C. The pellet was resuspended in 50 μL Buffer F with 0.5 μL RNase inhibitor (SUPERase In, Ambion), flash frozen, 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: GSM3666506