Role of Fun30 in chromatin remodelling and centromere function
Role of Fun30 in chromatin remodelling and centromere function
Total RNA for RNA-Seq was isolated using the hot acidic phenol method (Collart and Oliviero, 2001). Next 10 ug aliquots were treated with DNase I amplification grade (Invitrogen) for 30 minutes at 37C, purified by ethanol precipitation and quality-checked by 8M Urea 6% polyacrylamide gel electrophoresis in 0.5x TBE. PolyA RNA were purified using Oligo-dT Dynabeads (Invitrogen). The dynabeads selectively enrich poly-A RNA. Purified polyA RNA samples were concentrated by ethanol precipitation and then fragmented using the Ambion RNA fragmentation kit.  Samples were ethanol precipitated and the RNA was used in first strand and second strand cDNA synthesis with random hexamers at 150 ng/ ul. The entire reaction was used in library generation. 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 crosslink 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 adapterssynthesized 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_3_CATT_cDNA_FUN30
Experimental Factor: GENOTYPE
delta fun30
Experimental Factor: STRAIN_OR_LINE
W303a