title: GSM4066474: Wild type RNA-Seq replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: RNA preparation: Total RNA was prepared by homogenizing liver pieces in 10 volumes of Trizol reagent followed by the addition of 2 volumes of chlorophorm and centrifugation at 12000 g for 15 minutes. The aqueous phases were precipitated with isopropanol. Following resuspension in water RNAs were subjected to additional precipitation with ethanol. The RNA samples were digested with 10 units of DNase I for 10 min at 37 0C, followed by purification with phenol/chlorophorm extraction and ethanol precipitation. Chromatin immunoprecipitation: Liver tissue was minced to small pieces in PBS and after addition of formaldehyde to a 1% final concentration immediately was subjected to 10 strokes of dounce homogenization. Cross-linking was continued for 10 min and stopped by the addition of glycine at 0.125 M final concentration. Cross-linked cells were treated with a buffer containing 50 mM HEPES pH 7.5, 0.14 M NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40 and 0.25% Triton-X100, for 10 minutes and then with a buffer containing 0.2 M NaCl, 1 mM EDTA, 0.5 mM EGTA and 10 mM Tris-HCl pH 8.0 for 5 minutes. The nuclei were resuspended up to 4 ml with sonication buffer containing 10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate and 0.5% N-lauroylsarcosine and sonicated for 12 minutes in Covaris Sonicator instrument. After sonication, samples were supplemented with 400 μl of 10% Triton X-100 and centrifuged for 10 minutes at 20.000 rcf. After centrifugation, the extracted chromatin was incubated overnight with Dynabeads Protein G, that were prebound by 10 ug of the respective antibodies. The beads were washed six times with a buffer containing 50 mM Hepes pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, and 0.7% Na-deoxycholate, and then once with a buffer containing 20 mM Tris-HCl pH 7.6 and 150 mM NaCl. Immunoprecipitated chromatin was eluted from the beads and reverse crosslinked by overnight incubation with a buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1 % SDS, at 65 0C. Following incubation, samples were diluted by addition of one volume of TE. RNAs and proteins were removed by incubation with 20 ug/ml RNAse A for 30 minutes at 37 C, followed by incubation with 20 ug/ml Proteinase-K for 2 hours at 55 0C. DNA was extracted by phenol/chlorophorm and precipitated with ethanol. About 10 ng of the immunoprecipitated DNA and input DNA was used for library preparation. Libraries were prepared using TruSeq or NEBNext Adaptors, depending on whether less or more than 6 indexes were included. In all cases, 25 μl of immunoprecipitated DNA was purified using the MinElute columns (Qiagen, 28604) and eluted with 10 ul elution buffer. The concentration of the eluted DNA was measured in Qubit using the dsDNA HS Assay Kit (Invitrogen Q32851). Subsequently, ten ng of ChIP or input DNA were subject to end repair, in a mix containing T4 DNA polymerase (NEB M0203L), Klenow fragment (NEB M0210L), T4 DNA PNK (NEB M0201L) and dNTPs in T4 DNA Ligase Buffer (NEB, B02025) at 20 °C for 30 min. Following end repair, samples were purified using the Agentcourt AMPURE XP system (Agentcourt A63881) and subject to A addition by incubation with dATP and Klenow 3'-->5' exo- 37 °C for 30 min. Another AMPURE XP purification was performed and then Truseq or NEBNext adaptors were ligated using Quick Ligase in Quick Ligase Buffer. Following adaptor ligation, samples were incubated with 1.5 ul USER enzyme at 37 °C for 15 minutes, to cleave the adaptor hairpin loop. Purification and size selection was performed again by AMPURE XP and PCR amplification was performed using Truseq or NEBnext Primers in Kapa Hifi Hotstart mix. A final purification was performed and libraries were ready for Bioanalyzer assessment and sequencing.
attributes.tag: GEO Accession
attributes.value: GSM4066474