Illumina HiSeq 2000 paired end sequencing; ChIP-Seq of LET-418::3xFLAG in mixed stage early C.elegans embryos
ChIP-Seq of LET-418::3xFLAG in mixed stage early C.elegans embryos
Worms of the indicated phenotype were synchronised by bleaching and grown on EP+NA22 plates until L4 stage. L4 were then shifted to 25°C for 28h. Gravid adults were harvested and separated from laid eggs, bleached to harvest eggs within the animals.  Harvested embryos were washed with water until clear, and cross linked  30' at RT° in 2% PFA. Reaction was stopped with 125mM glycine for 20' at RT°. Embryonic pellets  were washed 2x in PBS 1x and snap-frozen until further use. Embryonic pellets were lysed in FA-150 (150mM NaCl, 50mM Hepes pH7.5, 1mM EDTA, 1% Triton X100, 0.1% sodium deoxycholate). sonication was performed with a Misonix 3000 microprobe for 10 cycles of 15'', output 5.0 and checked by DNA gel for 300-800bp fragments. Soluble lysates were then recovered by centrifugation. Immunoprecipitation (IP) was performed with 50ul ProteinG-Dynabeads (thermo) per IP and 4ug M2 anti-flag antibody (Sigma). A pre-clearing was performed with 20ul ProteinG-Dynabeads per IP tube prior to ab-specific IPs. IPs were washed 2x with FA-150, 2x with FA-500 (same recipe as FA-150 but with 500mM NaCl), 1x with FA-1000 (idem but with 1M NaCl), 1x with DOC buffer (0.25M LiCl, 1%NP-40, 1% sodium deoxycholate, 1mM EDTA pH8, 10mM Tris pH8), 2x with TE. IPs were then resuspended in TES (1xTE, 1%SDS) and de-crosslinked 0/n at 65°C. DNA was then extracted using the PCR purification kit (Qiagen).  Input DNA corresponds to 1% of the lysate used per IP and was not enriched for specific proteins, but was processed through de-crosslinking and DNA purification in parallel with the ChIP samples. DNA quality was assessed with a Bio-Analyser 2100 expert using the High sensitivity DNA assay (Agilent). DNA fragments were gel purified and processed for library preparation with the TruSeq ChIP sample kit (Illumina).
Experimental Factor: input DNA
immunoprecipitate