GSM1363114: Pc RNA Helicos; Drosophila melanogaster; RNA-Seq
Soluble chromatin was incubated with IgG agarose beads (Sigma, Cat # A2909). For every 10 ml of sonicated chromatin, 0.5-1 ml beads were added (for 50 ml of 3rd instar larvae or adult fly chromatin 1 ml beads was used). The mixture was rotated end-over end in the 15 ml Falcon tubes for 12-16 h at +4°C. Beads were then washed 3 times for 10 min at +4°C with 15 bead volumes of RIPA buffer and spun down for 5 min at 1000g, +4°C between washes. Beads were then washed for 10 min with 15 bead volumes of buffer TEN 140 buffer with 0.1% Triton X-100 at +25°C and spun down for 5 min at 1000 g, +25°C. In order to elute the complexes (protein-DNA-RNA), 12-15 bead volumes of IgG Elution buffer were added to the beads. The slurry was mixed by inversion for 1 hour at +25°C. This elution step was repeated one more time. In order to eliminate urea from the samples, the eluates were first concentrated  in 10K Amicon Ultra-15 columns (3000g, 15min at +25°C) and then washed/buffer exchanged 3 times with 15 ml of TEN 140 buffer with 0.1% Triton X100 (3000g, 5-15min at +25°C) in a fresh Amicon column. The resulting 0.5 ml concentrate was diluted with 2500 ul of RIPA buffer and transferred into two Eppendorf tubes.  Biotin-Streptavidin affinity purification   150-300 ul of Streptavidin Agarose beads (Thermo Scientific, cat # 20349) were added to each tube.  The mixture was rotated end-over-end for 12-16 hours at +16°C. Beads from both tubes were pooled into one 15 ml Falcon tube washed once with RIPA buffer, once with TEN 140 buffer with 0.1% Triton X100, twice with IgG Elution buffer and twice with IgG Elution buffer without SDS. For each washing step, beads were mixed with 12 ml of a given buffer on a rotating wheel for 10 min at +25°C and spun down for 5 min at 1000 g, +25°C. Beads were re-suspended in 10 ml of TEN 140 buffer and divided into three tubes: 6 ml for protein work, 2 ml for DNA and 2 ml for RNA analysis. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit. 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 12 cycles and library fragments from 300-700 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 following the manufacturer's
GEO Accession
GSM1363114