 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 23, 2016 |
| Title |
KC_cgRNAi_13_1 |
| Sample type |
SRA |
| |
|
| Source name |
KC167
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: KC167
cell type: Embryonic cells
lineage: Embryonic cells
conditions: cgRNAi
viewpoint: B1,X1,X2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
5X10^6 cells used to generate the primary 3C template. Cells were collected, centrifuged at 1200rpm, the supernatant was removed and are resuspended in PBS/10% FBS. Then the cells are cross-linked with formaldehyde to final concentration of 2% for 10min. The reaction was quenched by addition of glycine to final concentration of 0.125M. The cells were centrifuged and resuspended in cold lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100, 1 tbl Protease inhibitor). The nuclei are extracted and resuspended in 450µl UPW. For digestion, we add 60µl DpnII buffer, 15µl 10% SDS and incubate on a Thermomixer (Eppendorf) 37c, 60 minutes. 75µl of 20% TX-100 are added and the tube is incubated again 37c, 60 minutes. The chromatin is then digested with HC DpnII (NEB R0543M) in three rounds as following: 200U for 2 hours, 200U overnight and 200U for 2 hours. An aliquot is taken from the tube and run on gel to inspect digestion efficiency. The enzyme is then inactivated by incubating 65c, 20min. Next, the chromatin is ligated with 15µl of HC T4 ligase (NEB M0202M), with 10x ligase buffer to final volume of 7mL and incubated overnight at 16c. Aliquot is taken from the sample and ran on gel to inspect ligation efficiency. The chromatin is then reverse cross-linked overnight at 65c with 30ul proteinase K (Sigma-Aldrich), and then 30ul of RNase A are added and incubated for 45 minutes, 37c. The 3C DNA is then extracted by Ampure beads. Inverted head segments of 400 OR (wild type) or 200 cgKG000882/cg1 (mutant) animals were fixed in 2% formaldehyde in PBS (20 min., Room Temperature). Fixed tissue was then washed twice with PBS/125mM Glycine/0.01% Triton]X]100, and a third time with PBS. Samples were washed again with PBS/1% protease inhibitor cocktail (P8340,Sigma).Wing Imaginal discs were then dissected out and placed in an eppendorf tube. Excess fluid was removed and tissues were snap freezed in Liquid Nitrogen and stored at 80 C. To prepare DNA, discs were thawed on ice and centrifuged at 13,000 rpm for 20 sec. Excess fluid was discarded. Discs were resuspended in 50 ul lysis buffer (10 mM Tris]HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA360 (Sigma I8896)), and 10 ul/ml of protease Inhibitors (Sigma"P8340).Discs were homogenized with a plastic motorized pestle (3X2min) and continuation of the 4C protocol was as performed for KC cells.
5-10ug DNA in 200µl Elution Buffter (EB, 10mM Tris-HCl, pH 8) of 3C template is sonicated at 4°c using Bioruptor program: 5 cycles of 30s on, 60s off. The sonicated material is inspected by running 10µl on agarose gel to assure size distribution of 500-550 bps. The sonicated DNA is subjected to end-repair reaction - 20µl 10X end-repair buffer and 10µl end-repair mix (NEB E6050L) are added to the mix and incubated at 20°c for 30 minutes. The DNA is cleaned by x2.2 AmpureXP beads (Beckman Coulter) and eluted in 76µl EB. Two nested PCR reactions are used for UMI-4C library construction. The PCR reactions are performed in 50ul volume with the following reagents: 10ul GoTaq Flexi buffer (promga M792A), 3ul MgCl 25mM (promega A3511), 0.2mM dNTPs, 2µl 10mM bait primer (0.4mM final concentration), 2µl 10mM Illumina enrichment primer 2 (0.4mM final concentration), 1ul GoTaq (promga M3008) and 200ng UMI-4C DNA template. PCR program is 2 minutes 95°c, 20 cycles of 30s 95°c, 30s 56°c, 60s 72°c, final extension of 5min 72°c. Following the PCR reaction we clean the amplified DNA with x1 SPRI beads and use 75% of the eluate to a second PCR reaction. The second PCR reaction is identical to the first. The only difference is the use of the bait primer with the Illumina dangling adapter (As shown in figure 1B). In the second PCR reaction 15-18 cycles are used instead of 20. Then, A-tailing reaction is performed with 10µl NEBbuffer2, 4µl Klenow (NEB M0212M), and 10µl 10nM dATP. Following A-tailing, the 5’ ends of the DNA are dephosphorylated with 2µl of Alkaline Phosphatase (NEB M0290S) 50°c for 60 minutes, and the DNA is cleaned with AmpureXP beads (x2) and eluted in 67µl EB. The DNA template is ligated with 5µl 15µM Illumina indexed adapters (final concentration 0.4µM), 10µl ligase mix and 80µl 10X buffer (NEB M2200). To release the non-ligated strand of the adapter, the DNA is denatured at 95°c for 2 minutes and cleaned with SPRI beads x1.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
The data was processed using UMI-4C pipeline (https://bitbucket.org/tanaylab/umi4cpackage). With default parameters.
Processed reads were mapped with bowtie2 with the parameters: --reorder --end-to-end.
Genome_build: dm6
Supplementary_files_format_and_content: The processed adj files are a 4 columns tab delimited table with headers: viewpoint (bait), fragment end id1 (fend1), fragment end id2 (fend2), UMI count (count)
|
| |
|
| Submission date |
Sep 22, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Lilach Gilboa |
| E-mail(s) |
[email protected]
|
| Phone |
+97289343794
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Biological Regulation
|
| Lab |
Room 326
|
| Street address |
234 Herzl Street,
|
| City |
Rehovot |
| ZIP/Postal code |
7610001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL19132 |
| Series (1) |
| GSE87209 |
UMI-4C for quantitative and targeted chromosomal contact profiling |
|
| Relations |
| BioSample |
SAMN05804865 |
| SRA |
SRX2185114 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2325208_KC_cgRNAi_13_1.adj.txt.gz |
113.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
