GSM3537042: Vitamin D input; Homo sapiens; ChIP-Seq
Cross-linked using 1% formaldehyde for 10 min at RT, then 125 mM glycine for 5 min. Plates were washed 2X with ice-cold PBS, incubated in TrypLE at 37˚C for 3 min, and washed 2X with ice-cold PBS. Cells were lysed in Nuclear Isolation Buffer from Magna ChIP HiSens Chromatin Immunoprecipitation Kit (Millipore) on ice for 20 min with vortexing for 10 s every 5 min. Nuclei were pelleted at 800xg 5 min, then re-suspended in 1 ml RIPA buffer with Protease Inhibitor Cocktail III (Millipore). Cells were sonicated on ice for 15 s 9X setting 2 on a Sonic Dismembrator model 100 (Thermo Fisher). 10 µl of sonicated DNA (~10 ng) was saved as input control. 1ml of sonicated DNA (~1 µg) was used for VDR and normal IgG ChIPs. 300 µl Dynabeads (Thermo Fisher) were washed twice with 1.5 mL 1 mg/ml BSA/PBS 2 mM EDTA solution and once with 1.5 ml 5 mg/ml BSA/PBS. Beads were re-suspended in 1000 µl with 1 mg/ml BSA/PBS 2 mM EDTA solution and incubated with 24 µg of anti-VDR C-20x sc-1008x rabbit polyclonal (Santa Cruz) for 5 hrs, then washed again as above. Antibody-conjugated beads were incubated with sonicated DNA overnight rotating at 4˚C. Beads were washed 1x with 5:1 RIPA:PBS containing 5mg/mL BSA (fraction V) and Protease Inhibitor Cocktail III (Millipore), then 5x in LiCl IP wash buffer (100 mM Tris-HCl pH 7.5/ 500 mM LiCl/1% NP-40 /1% Sodium deoxycholate) and 1x in TE buffer. Bound DNA was eluted off the beads twice, with 100 µl ChIP Elution Buffer (Millipore) containing 283 mM NaCl and 1 µl proteinase K and heated at 65˚C for 1 hr with vortexing every 15 min. Eluted samples were combined and incubated at 65˚C overnight. Input 10 µl sample was combined with 90 µl ChIP Elution Buffer (Millipore) and 283 mM NaCl and 1 µl proteinase K and incubated at 65˚C along with the other samples. DNA was purified using the Clontech NucleoSpin kit (Clontech) and eluted twice using 20 µl of 70˚C elution buffer. Libraries were prepared from 10 ng of ChIP DNA with the Library Construction Kit Kapa Biosystems (Wilmington, MA) with two modifications: adaptors were diluted 1:20 and adaptor-modifed DNA were amplified for 10 cycles. The pooled libraries were sequenced on one lane each for 101 cycles on a HiSeq2500 in High Output mode using a TruSeq SBS sequencing kit version 3.
GEO
GSM3537042
GEO Accession
GSM3537042