title: GSM5210225: Adrenal_Glands_KO_ChIP_1; Mus musculus; ChIP-Seq
library_construction_protocol: The adrenal glands from 10-14-month old WT and KO female mice were resected and stored in liquid nitrogen immediately until use. Each gland was homogenized in 200 µl ice-cold PBS buffer with a handheld Polytron homogenizer. The final volume was brought to 1 ml with ice-cold PBS. The homogenized samples were crosslinked with 66.7 µl 16% formaldehyde (1% final) at room temperature for 15 minutes. They were quenched with 107 µl 1.25 M glycine (0.125 M final) at room temperature for another 10 minutes in rotating tubes. The samples were centrifuged, and the pellets were washed twice with ice-cold PBS. The crosslinked pellets were re-homogenized with the Polytron homogenizer and suspended in 300 µl swelling buffer (50 mM Tris (pH 8.0), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and incubated on ice for 20 minutes to release nuclei. The nuclei were harvested by centrifugation, resuspended in 200 µl ChIP sonication buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 0.5% SDS), and incubated on ice for 20 minutes. The nuclei were sonicated with a probe-based sonicator (FB120, probe:CL-18, FISHER SCIENTIFIC) at a 20% amplitude setting. The sonication was conducted using 15-second pulses at 15-second intervals for a total of 8 minutes. The sonicated nuclei were harvested by centrifugation, and then diluted with 800 µl ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris (pH 8.1), 167 mM NaCl) to reach the final SDS concentration of 0.1%. These samples represented sonicated chromatin ready for immunoprecipitation.  To quantify chromatin and assess the degree of its fragmentation, we treated 5% of the sonicated nuclei (50 µl/sample) with 10 µg of RNase A for 15 minutes at 37 ºC followed by 20 µg of proteinase K for 30 minutes at 65 ºC. They were de-crosslinked for 5 minutes at 95 ºC. DNA was extracted with the QIAquick PCR Purification Kit. DNA concentration was determined with a Nanodrop 1000 Fluorospectrometer. DNA fragment sizes were confirmed to be 200-800 bp in length, according to electrophoresis.  For immunoprecipitation, an equal amount of sonicated chromatin, based on their prior DNA measurements, of different samples was reacted with anti-RPB1 N-terminal domain Ab (D8L4Y) (1:100) at 4 ºC overnight, followed by 40-µl magnetic protein G beads (Bio-Rad) for another 2 hours at 4 ºC. The beads were rinsed with wash buffer (100 mM Tris (pH 8.0), 500 mM LiCl, 1% NP-40, 1% deoxycholic acid) for 5 times and then with TE buffer once. The chromatin was eluted with elution buffer (50 mM Tris (pH 8.0), 10 mM EDTA, 1% SDS) at 65 ºC for 10 minutes. The immunoprecipitated chromatins were de-crosslinked at 65 ºC overnight with NaCl adjusted to 200 mM. The chromatins were then treated with 10 ug RNase A/sample at 37 ºC for 1 hour, followed by 200 g proteinase K/sample for 2 hours at 45 ºC. DNA of the samples was purified with QIAquick PCR Purification kit and quantified by the Bioanalyzer (Agilient). Libraries were prepared robotically with 0.2 to 2 ng of fragmented DNA ranging 100-300 bp in length, using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs), as per the manufacturer's recommendations. Adapters and PCR primers were purchased from Integrated DNA Technologies . Size selection was carried out using SparQ beads (Qiagen) prior to PCR amplification (12 cycles). Libraries were quantified using the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems). Average size fragment was determined using a LabChip GX (PerkinElmer) instrument. The libraries were normalized and pooled, and then denatured in 0.05 N NaOH and neutralized using HT1 buffer. The pool was loaded at 225 pM on an Illumina NovaSeq S4 lane using Xp protocol as per the manufacturer's recommendations. The run was performed for 2 x 100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at 1% level. Each library was sequenced at 25 million reads.