title: GSM1936638: Naive_hmC_medIP-Seq_7_12; Homo sapiens; MeDIP-Seq
library_construction_protocol: To generate nuclear extracts, naïve and activated CD4+ T cells were pelleted, washed once in cold PBS, resuspended in 100 µl hypotonic lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA) supplemented with 2 mM Na3VO4 and 1x Halt protease inhibitor cocktail (Thermo Scientific), and placed on ice for 10 minutes. NP-40 was then added to a final concentration of 0.4% and vortexed vigorously for 15 seconds. Samples were centrifuged for 6 minutes at 3300 x g at 4oC and pellets were resuspended in 60 µl nuclear lysis buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 10% glycerol) supplemented with 2 mM Na3VO4 and 1x Halt protease inhibitor cocktail. Lysates were passed through a 23 gauge needle 4 times and left on ice for 15 minutes. After vortexing for 15 seconds, samples were centrifuged at maximum speed (20,000 x g) for 5 minutes at 4oC and supernatants containing nuclear proteins were transferred to fresh tubes. Protein concentrations were determined with the Pierce BCA Protein Assay (Thermo Scientific). 7.5-15 ug of nuclear lysates were resolved with a 3-8% NuPage Tris-Acetate gel (Invitrogen) and transferred to PVDF. Sequencing libraries were constructed from DNA samples with the Illumina TruSeq V3 library construction protocol.
attributes.tag: GEO Accession
attributes.value: GSM1936638