title: GSM1701777: RAG2ko_thy_mm_TCRBetaTg_RNAseq C22; Mus musculus; MNase-Seq
library_construction_protocol: Thymocytes were crosslinked for 15 minutes with 1% formaldehyde, and the crosslinking reaction was quenched with 0.125 M glycine.  Cells were washed twice with ice-cold PBS containing 1 mM PMSF and 1 µg/mL pepstatin A.  Five million crosslinked cells were resuspended in MNase digestion buffer (50 mM Tris-HCl, pH 7.6; 1 mM CaCl2, 0.2% Triton X, 5 mM sodium butyrate, 1 mM PMSF, 1 µg/mL pepstatin A).  Micrococcal nuclease (MNase, 0.2 U) was added to the sample, which was incubated at 25 ºC for 5 minutes.  The nuclease digestion was terminated with 5 mM EDTA.  Crosslinks were reversed using an overnight incubation at 65 ºC in the presence of 0.5% SDS and 200 µg/mL Proteinase K.  DNA was isolated by standard phenol:chloroform extraction and ethanol precipitation.  The sample was then subjected to RNAse digestion (10 µg/mL RNAse A at 37 ºC for 1 hour) and a second proteolysis step to remove the RNAse (200 µg/mL Proteinase K, 0.3% SDS, 65 ºC, 1 hour).  Again, the DNA was isolated by phenol:chloroform extraction and ethanol precipitation and resuspended in water.  The sample was run on a 2% agarose gel, and the ~150 bp mononucleosome band was excised and purified using the Qiagen Gel Extraction kit. libraries were prepared and sequenced following standard Illumina protocols
identifiers.namespace: GEO
identifiers.id: GSM1701777