title: Illumina HiSeq 2000 sequencing; Mitochondrial heteroplasmy in vertebrates using ChIP-sequencing data
design: Mitochondrial heteroplasmy in vertebrates using ChIP-sequencing data
library_construction_protocol: For fresh specimens, hepatocytes were prepared by direct perfusion of the liver with buffered salt solution, followed by cross-linking of the diced tissue in 1% formaldehyde solution for 20 minutes, addition of 250 mM glycine and incubation for a further 10 minutes to neutralize the formaldehyde. Liver samples from frozen specimens were powdered while frozen by using a mortar and pestle on dry-ice, and the powdered frozen tissue was subsequently cross-linked as described above. After homogenization of cross-linked liver tissue in a dounce tissue grinder, hepatocytes were rinsed with PBS and lysed according to published protocols {Schmidt, 2009} to solubilize DNA-protein complexes.  Chromatin was fragmented to 300 bp average size by sonication on a misonix sonicator 3000 with a 418 tip.  Chromatin from 0.1 g of dounced liver tissue was used for each ChIP experiment using antibodies against H3K4me3 (millipore  05-1339), H3K27ac (abcam ab4729), H3K4me1 (abcam ab8895) or total histone H3 (abcam  ab1791),  and automated 96-well protocols in an Agilent Bravo liquid handling robot {Aldridge, 2013}. Step 1: Attachment of antibody to Protein G beads. 25μl of Protein G Dynabeads (Invitrogen) per ChIP were washed with a 0.5%BSA/PBS solution followed by addition of 2.5 (H3K4me3, total histone H3) or 5μg (H3K27ac, H3K4me1) of antibody. The plate was sealed and transferred to 4oC and mixed for a minimum of four hours on an orbital shaker (Grant-bio, PMS1000). Step 2: Wash and addition of lysate. Antibody bound protein G beads were washed in 0.5%BSA/PBS solution and 180 μl of the sonicated lysates were added to the prepared beads. The plate was returned to a 4C cold room for overnight immunoprecipitation. Step 3: Wash of DNA bound beads. ChIP-DNA bound beads were washed ten times in 180 μl cold RIPA solution, and immunoprecipitated DNA was eluted in 50 μl elution buffer and transferred to rigid PCR 96-well plates for crosslink-reversal in a 65C thermal cycle for a minimum of five hours. Step 4: Removal of beads, RNase and Proteinase K treatment. 50 μl of TE was added to beads to dilute SDS in elution buffer. 2 μl RNase (Invitrogen, AM2269) was added to eluted ChIP-DNA and incubated on Bravo deck at 37C for 30 minutes, followed by  2 μl of Proteinase K treatment (Invitrogen, AM2548) at 55C for 1-2 hours. Step 5: Purification of DNA. Phenol and Ethanol precipitation was replaced with an Ampure Bead (Beckman Coulter, A63881) clean up step. 180μl of SPRI beads (1.8 times volume) were added to the DNA followed by two 70% ethanol washes. DNA eluted in 50 μl EB buffer was used for library preparation. CEBPA chromatin immunoprecipitations were carried out with a manual version of the above protocol, using 0.1-0.5g of liver tissue and 10 ug of antibody sc-9314 (Santa Cruz Biotechnology). Illumina sequencing libraries were prepared from ChIP-enriched DNA in 96 well microtitre plates using automated liquid handling robotic platforms. Pre-PCR library preparation steps were carried out  using a Beckman Fxp dual arm instrument with cytomat microplate hotel. Briefly, 50μl of ChIP DNA was purified by binding to twice the volume of AMPure XP beads (Beckman Coulter, Inc.) and eluted in 30μl of 10mM Tris-HCl, pH8.5. End-repair, A-tailing and paired-end adapter ligation were performed using NEBnext reagents from New England Biolabs (E6000S), with purification using a 1:1 ratio of AMPure XP to sample between each reaction. Illumina adapters were used at a final concentration of 20pM (a 1:20 dilution of our standard library adapter concentration) to reduce adapter dimer formation. After ligation, excess adapters and adapter dimers were removed using two Ampure XP clean-ups, first with a 0.7:1 ratio of standard Ampure XP to sample, followed by a 1:1 ratio, with elution in eluted in 30μl of 10mM Tris-HCl, pH8.5. 10μl of this adapter ligated material was then used as template for PCR amplification with Kapa HiFi 2 x mastermix (Kapa Biosystems KK2602) with 200nM final concentration of standard PE1.0 and modified multiplexing PE2.0 primers. After PCR setup on the Beckman Fxp , PCR reactions were cycled on an MJ Tetrad thermal cycler with the following conditions: 94°C -2 minutes; 10-15 cycles of  94°C -20 seconds, 65°C -30 seconds, 72°C -30 seconds; 72°C - 3 minutes. 10 cycles were used input DNA (500 ng) and 15 cycles for ChIP DNA. After PCR, excess primers and any primer dimers were removed by performing a 0.7:1 Ampure XP clean-up on a Caliper Zephyr liquid handler with elution in 30μl of 10mM Tris-HCl, pH8.5. Libraries were pooled in equal volume and the concentration of that pool determined  by real-time PCR using the SYBR Fast Illumina Library Quantification Kit (Kapa Biosystems) before sequencing on an Illumina MiSeq, 50 cycles single end plus index read,  to determine the relative representation of each barcoded library. Based on this data the library pool was reblended so as to give equal representation of each library, and requantified by real time PCR as above, before sequencing on an Illumina HiSeq 2000 for 50 cycles single end, plus index read.
attributes.tag: Experimental Factor: H3K27Ac
attributes.value: immunoprecipitate
attributes.tag: Experimental Factor: Monodelphis domestica
attributes.value: organism
attributes.tag: Experimental Factor: juvenile
attributes.value: developmental stage
attributes.tag: Experimental Factor: male
attributes.value: sex