title: GSM1696982: Sample 11: invitro2_3ugOctamer_lightdig_Rep1; Tetrahymena thermophila; MNase-Seq
library_construction_protocol: Preparations were performed as described (Jacob et al. 2004), with minor modifications. Log-phase or starved cells were centrifuged at 1000 g for 5 min, and resuspended in 70 ml TMS (10 mM Tris pH 7.5, 10 mM MgCl2, 3 mM CaCl2, 0.25 M sucrose, 1 mM DTT, 0.16 % [v/v] NP40). The culture was lysed in a Waring PBB212 Blender at the “High” setting for 25 sec, as previously described (Gorovsky et al. 1975). Formaldehyde was simultaneously added to a final concentration of 1% (v/v), at the onset of blending. The resulting cell lysates were stirred on ice for 15 min before quenching with 125 mM glycine, and subsequent stirring on ice for an additional 10 min. These fixation and quenching steps were omitted in experiments to prepare native chromatin. Sucrose was then added at 0.816 g per ml lysate, with constant stirring on ice for 10 min. Upon complete dissolution of sucrose, the lysates were cleared by centrifugation at 9000 g for 30 min. The resulting macronuclear pellet was washed in buffer A (15 mM Tris pH 7.5, 60 mM KCl, 15 mM NaCl, 2 mM CaCl2, 0.5 mM spermidine trihydrochloride, 0.15 mM spermine tetrahydrochloride, 1 mM DTT), centrifuged at 1200 g for 2 min, and resuspended in 1ml buffer A. An 830 μl aliquot of macronuclei was pre-incubated at 37 oC for 5 min. From this, 100 μl macronuclei was withdrawn and mixed with 52 μl lysis buffer (300 mM NaCl, 30 mM Tris pH 8, 75 mM EDTA, 1.5 % (w/v) SDS, 1.5 mg/ml Proteinase K) to serve as an undigested control. Subsequently, 2,000 Kunitz units of MNase (NEB) were added to the macronuclei, and incubated at 37oC for 45s, 1 min 15 s, 2 min 30 s, 5 min, 7 min 30s, 10min, and 15 min respectively. At each time point, 100 μl macronuclei was withdrawn and mixed with 52 μl lysis buffer. All samples were incubated at 65 oC overnight to reverse formaldehyde crosslinks and digest proteins. DNA was subsequently purified through phenol-chloroform extraction, then ethanol-precipitated, and resuspended in buffer EB (Qiagen). 1μl of DNA from each sample was run on a 2 % agarose-TAE gel to check the progression of MNase digestion. The sample with ~80 % mononucleosomal and ~20 % dinucleosomal DNA (Supplemental Fig. S3) was deemed “light digest”. This is in accordance with previous recommendations for an adequate level of MNase digestion in nucleosome mapping studies (Zhang and Pugh 2011). Separately, the sample exhibiting almost exclusively mononucleosomal DNA with a significant smear in the subnucleosomal region (Supplemental Fig. S3) was deemed “heavy digest”. Light and heavy-digest samples were run on a 2 % agarose-TAE gel, and the mononucleosome-sized fragment was excised and purified using a QIAquick gel extraction kit (Qiagen). Illumina libraries were prepared from mononucleosomal DNA according to manufacturer’s instructions, and subject to paired-read sequencing. Reconstituted chromatin was adjusted to 5 mM MgCl2, 5 mM CaCl2, 70 mM KCl, and 10 mM HEPES pH 7.9. For in vitro experiments #1, the chromatin was digested with 22 Kunitz units of MNase (NEB), at 25 oC for 12 min. The digestion reaction was stopped by the addition of 21.6 μl buffer E (33 mM Tris pH 8, 100mM EDTA, 0.67 % (w/v) SDS, 16.7 % (v/v) glycerol) and 8.4 μl 20 mg/ml proteinase K. For in vitro experiments #2, all of the reagent volumes were tripled. The amount of MNase was doubled further, for the heavy digest sample. Samples were incubated at 50 oC at 1 hr. and then loaded on a 2 % agarose-TAE gel. The mononucleosome-sized fragment was gel-purified using a QIAquick gel extraction kit (Qiagen). Illumina libraries were then prepared from mononucleosomal DNA according to manufacturer’s instructions. 12.9 μg macronuclear gDNA was made up to 200 ul with TMS, and then digested with 1.79 Kunitz units of MNase (NEB) for 7 min at 25 oC. The reaction was terminated by adding 112 μl stop buffer (300 mM NaCl, 30 mM Tris pH8, 75 mM EDTA, 1.5 % [w/v] SDS, 1.5 mg/ml Proteinase K), and subsequently purified through phenol-chloroform extraction and ethanol precipitation. MNase-digested gDNA was resuspended in 25 μl buffer EB (Qiagen) and loaded on a 2 % agarose-TAE gel, and the mononucleosome-sized fragment was gel-purified using a QIAquick gel extraction kit (Qiagen). Standard TruSeq Illumina library construction protocol
attributes.tag: GEO Accession
attributes.value: GSM1696982