7 protocols
AccessionNameType
P-MTAB-26274
bioassay_data_transformation
Reads were mapped to the Drosophila reference genome (version 5.30). Up to 2 mismatches in each read were allowed; non-unique reads were discarded. Options for bowtie were: -S –p 20 –v 2 --tryhard –p 24 –m 1 --best --strata
P-MTAB-26273
scanning
Base calls were done by the Illumina software pipeline using RTA 1.12.4.2 and BCL converted using CASAVA 1.8.2 (configureBclToFastq.pl).
P-MTAB-26272
sequencing
Single-end sequencing; according to manufacturers protocols
P-MTAB-26270
nucleic_acid_extraction
30 ml cell suspension were spun down and the cell pellet was resuspended in 1 ml of cross-linking solution (50 mM HEPES, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). 37% formaldehyde was added to a final concentration of 1.8%. The tube was rotated at room temperature for 15 minutes and the reaction was quenched with 2.5 M glycine at a final concentration of 125 mM for 5 minutes. The fixed cells were washed 3 times for 5 minutes each with rinse buffer 1 (10 mM HEPES pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) and 5 times for 5 minutes each with rinse buffer 2 (10 mM HEPES pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). Finally, the cells were washed 3 times in RIPA buffer (25 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton-X 100, 0.1% SDS, 0.1% DOC). The cells were sonicated 30 x 20 seconds using a Branson 250 sonicator at pulse 40, intensity 3%, followed by Covaris sonication. The chromatin should be sheared into fragments with average length of 200 bp. Samples were centrifuged at maximum speed and the supernatant was used for the subsequent steps. To reverse cross-linking, samples were incubated at 65C overnight in TE buffer, followed by a treatment with RNaseA (0.2 mg/ml) for 30 min at 37C, and with Proteinase K (0.05 mg/ml) for 2 hours at 50C. Finally, DNA was purified using Minelute columns (Qiagen).
P-MTAB-26269
treatment
Cells at a density of 1 million per ml were transfected with 10 ug dsRNA against NSL1, NSL3 or GFP using Lipofectamine RNAiMAX (Invitrogen) according to manufacturers instructions. The cells were harvested after 6 days.
P-MTAB-26271
nucleic_acid_extraction
30 ml cell suspension were spun down and the cell pellet was resuspended in 1 ml of cross-linking solution (50 mM HEPES, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). 37% formaldehyde was added to a final concentration of 1.8%. The tube was rotated at room temperature for 15 minutes and the reaction was quenched with 2.5 M glycine at a final concentration of 125 mM for 5 minutes. The fixed cells were washed 3 times for 5 minutes each with rinse buffer 1 (10 mM HEPES pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) and 5 times for 5 minutes each with rinse buffer 2 (10 mM HEPES pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). Finally, the cells were washed 3 times in RIPA buffer (25 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton-X 100, 0.1% SDS, 0.1% DOC). The cells were sonicated 30 x 20 seconds using a Branson 250 sonicator at pulse 40, intensity 3%, followed by Covaris sonication. The chromatin should be sheared into fragments with average length of 200 bp. Samples were centrifuged at maximum speed and the supernatant was used for the subsequent steps. 3 ul antibody were added to the chromatin and rotated overnight at 4C. Protein A/G-Sepharose was blocked with 0.1% BSA and 1 mg/ml of short dsDNA (15 bp) for 1 hour. 20 ul of the sepharose was added to the chromatin/antibody mixture and incubated for 3 hours in 4C. The sepharose beads were then washed four times for 15 minutes in RIPA buffer, once in LiCl buffer (0.25 M LiCl, 10 mM Tris at pH 8, 1 mM EDTA, 0.5% NP-40, 0.5% DOC), and two times in TE buffer. The beads were incubated at 65C overnight in TE buffer to reverse the cross-link. The sample was then treated with RNaseA (0.2 mg/ml) for 30 min at 37C, and with Proteinase K (0.05 mg/ml) for 2 hours at 50C. Finally, DNA was purified using Minelute columns (Qiagen).
P-MTAB-26268
grow
S2 cells were grown in Schneiders Media (Invitrogen) supplemented with 10% of heat-inactivated bovine fetal serum to a density of 1 million cells per ml.