Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: After removal from jelly, eggs were fixed in 2% formaldehyde for 12 minutes at room temperature. Cross-linking was stopped by adding 125mM glycine followed by rotation for 10 min at 4 degrees. After washing with cold PBS and HEG Buffer (50mM HEPES pH7.5, 1mM EDTA, 20% Glycerol), embryos were snap-frozen and stored at -80 degrees. Embryos were homogenized in Nuclei Buffer 1 (50mM HEPES pH7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton X-100, 1mM DTT) and centrifuged at 500g for 3 minutes, after which the pellet containing debris was discarded. After another centrifugation at 2000g for 10 minutes the pellet contained nuclei, which were washed with Nuclei Buffer 2 (50mM HEPES pH7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton X-100) and finally resuspended in Lysis Buffer (50mM HEPES pH7.5, 500mM NaCl, 1mM EDTA, 0.1% DOC, 01% Triton X-100, 0.1% SDS). After shearing the chromatin to 100-200bp using a Covaris Instrument and removing the non-soluble material, we blocked the chromatin with pre-blocked (1mg/ml tRNA, 1mg/ml BSA) protein A beads CL-4B (GE Healthcare 71-7090-00 AE). At this point we removed a small aliquot of chromatin for Input DNA and incubated the remaining chromatin with the desired antibodies, rotating at 4 degrees over night. We then added pre-blocked protein A beads to bind the antibodies and washed the beads twice with Lysis Buffer, twice with DOC Buffer (10mM Tris pH8, 0.25M LiCl, 0.5% NP-40, 0.5% DOC, 1mM EDTA), and once with TE Buffer. Chromatin was eluted from the beads with Elution Buffer (1%SDS, 0.1M NaHCO3) and de-crosslinked. RNA was isolated using Trizol. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 18 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer or HiSeq machine following the manufacturer's protocols. Strand-specific RNA-seq libraries were prepared using dUTP incorporation during the 2nd strand synthesis.