Role of Fun30 in chromatin remodelling and centromere function
Role of Fun30 in chromatin remodelling and centromere function
Input libraries were created from crosslinked chromatin before chromatin immunoprecipitation. ChIP was carried out essentially as described (Neves-Costa et al., 2009).  Briefly, samples were crosslinked 60 min with 1 % final formaldehyde and chromatin extracts were sonicated to 500 bp. Triplicates or duplicate ChIP samples were validated by qPCR. Illumina sequencing was performed using protocols derived from (Lefrancois et al., 2009; Marioni et al., 2008; Quail et al., 2009) and the standard Illumina protocol according the manufacturer. DNA fragments were purified after reversal of the crosslionk and concentrated using MinElute columns (QIAGEN). Eluted DNAs from two biological replicas (equal amount of DNA) were separated by electrophoresis through 2% agarose in TAE  and DNA fragments with size range 150-450 bp were excised. Excised DNA fragment were purified using the QIAGEN Gel Extraction Kit and eluted in 30 ml of EB buffer (10 mM Tris-HCL, pH 8). The entire size selected DNA was then used in the end-filling and A-tailing reactions, essentially as described in the standard Illumina protocol, using standard molecular biology reagents purchased from New England Biolabs. The adapter ligation step was performed using barcoded single-end adapters synthesized by Sigma-Genosys described in (Lefrancois et al., 2009). Briefly, forward and reverse adapters were mixed in equimolar ratios, incubated at 95C for 5 minutes, and allowed to anneal by using a ramp of -1C/ 10 seconds until the sample reached 4C. The ligation of adapters with DNA fragments was performed using T4 DNA ligase from Enzymatics, with incubation for 30 minute at 16C followed by an additional 30 minutes at 22C. Next the library was purified using the QIAGEN MinElute kit and separated in a 2 % agarose/ TAE gel for 1 hour at 90 V. Libraries were excised from the gel between 150 bp and 500 bp (always superior 110bp to avoid the risk of adapter dimer contamination). Amplification was performed using Pfx Platinum polymerase (Invitrogen) for 15 cycles as described by Quail et al. (Quail et al., 2009). The libraries were concentrated with QIAGEN MinElute kits and electrophoresed in a 1.9 % agarose/ TAE gel. Samples were quantified with SYBR Green qPCR Master Mix (Applied Biosystems) and the primers SYBR FP4 and SYBR RP7 and compared to a standard curve of phiX174 library, described by Quail, Swerdlow, and Turner (2009). The libraries were diluted to 10 nM in EB buffer.
Experimental Factor: INDIVIDUAL
s_4_ACGT_Input_FUN30
Experimental Factor: GENOTYPE
delta fun30
Experimental Factor: STRAIN_OR_LINE
W303a