title: Notch-GFP_W3L_ChIP-seq_ChIP_Rep2

description: ChIP DNA; Antibody information listed below: official name: GFP ab290;target name: Green Fluorescent Protein;host: Rabbit;antigen: entire GFP sequence;clonal: Polyclonal;purified: Crude;company: Abcam;catalog: ab290;reference: abcam website Jungreis et al. Genome Res. 2011 Dec;21(12):2096-113.;short description: This antibody is reactive against all variants of Aequorea victoria GFP such as S65T-GFP RS-GFP YFP and EGFP.

extract_protocol:
1. y; bw cn sp flies are cultivated in standard fly food covered with yeast powder.2. Flies are removed from the bottles after 24 hours and eggs are allowed to grow at 25 C for several days until they reach the desired growth stage.3. Wandering third instal larvae are collected from the side of the bottles using a brush. They are rinsed with EWB and cross-linked in 1.8% formaldehyde at room temperature for 15 min.4. Chromatin collection is performed as described in The ChIP-chip protocol from the White lab.
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911).  After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.

value: Notch-GFP

value: Unknown

value: Notch genomic coding region


title: seq-Ab52946_H3K14ac_FEM2_AD_Input_Rep1

status: Public on Aug 28 2013

type: SRA

city: Toronto

first: DCC

last: modENCODE

state: Ontario

address: MaRS Centre, South Tower, 101 College Street, Suite 800

country: Canada

institute: Ontario Institute for Cancer Research

description: input DNA

library_source: genomic

data_processing:
ChIP-seq_alignment-BWA:JL:1 protocol. BWA is a fast light-weighted tool that aligns relatively short sequences to a sequence database. The algorithms is based on Burrows-Wheeler Transform (BWT). This protocal, was ran as DEFAULT for both bwa aln and bwa samse.bwa aln: Find the SA coordinates of the input reads. Maximum maxSeedDiff differences are allowed in the first seedLen subsequence and maximum maxDiff differences are allowed in the whole sequence.OPTIONS:-n NUM
Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]-o INT
Maximum number of gap opens [1]-e INT
Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]-d INT
Disallow a long deletion within INT bp towards the 3?-end [16]-i INT
Disallow an indel within INT bp towards the ends [5]-l INT
Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ?-k 2?. [inf]-k INT
Maximum edit distance in the seed [2]-t INT
Number of threads (multi-threading mode) [1]-M INT
Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]-O INT
Gap open penalty [11]-E INT
Gap extension penalty [4]-R INT
Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).-c
Reverse query but not complement it, which is required for alignment in the color space.-N
Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.-q INT
Parameter for read trimming. BWA trims a read down to argmax_x{\sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l is the original read length. [0] bwa samse:Generate alignments in the SAM format given single-end reads. Repetitive hits will be randomly chosen.OPTIONS:-n INT
Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]  Processed data are obtained using following parameters: genome version is ce10

molecule: genomic DNA

organism: Caenorhabditis elegans

growth_protocol: Worm_adult_growth_and_harvest_vHP2.  Synchronized cultures of worms were grown until gravid in 400 mL batches.  Gravid adults were isolated through sucrose floatation, and frozen into pellets in liquid nitrogen with EBS (250 mM HEPES (pH 7.5), 1180 mM NaCl, 480 mM KCl, 20 mM EDTA, 5 mM EGTA, sucrose (final 340 mM) and protease/phosphatase inhibitors.  The pellets were stored at -80°C.

extract_protocol:
Worm extract was made from adult worms.  Frozen worm popcorn was ground up in a mixer mill.  Samples were then fixed with 1% formaldehyde for 10 minutes. Chromatin was then sheared by sonication in the Bioruptor, and the soluble fraction was collected and stored at -80°C for future use.
Worm extract from adult worms and 5 ?g of affinity purified antibody was used for ChIP. Dynal Protein A or G beads were used to recover the ChIPed DNA.  Samples were then treated with RNase A and Proteinase K, and reverse crosslinked at 65°C. ChIPed DNA was purified and then quantified using the Pico Green HS fluorescent assay.
Worm_ChIP-seq_ DNA_Library_Preparation_vSS2. ChIP and 10ng of input are blunted (T4 polymerase), an A-overhang is added (Klenow), DNA is ligated (concentrated T4 ligase) to annealed adaptors, size-selected, and amplified by PCR.Changes: Ligate O/N at 16C. Use adapters and primer cocktail from Illumina TruSeq kit. Use AMPure cleanup in the place of Qiagne cleanup. Use Pippin prep for size selection .
Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.

tag: strain

tag: developmental stage

value: Germline containing young adult

tag: genotype

tag: Sex

value: Hermaphrodite


title: seq-UP07442_H3K9me3_DAM1411287_N2_Eemb_Input_Rep1

description: input DNA

growth_protocol: Worm_embryo_growth_and_harvest_vSS5. Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Live embryos were cross-linked in M9 + 1.85% formaldehyde for 30 minutes at room temperature. Embryos were then washed twice with M9 Buffer, once with 100 mM Tris-HCl pH 7.5 and once with FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl). Pellets were frozen at -80C. For a detailed protocol see http://www.modencode.org/.

extract_protocol:
Worm_embryo_extraction_vSS2. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl) + protease inhibitors. Sample was dounced 30 times using tight pestle at 4ºC, then sonicated 9 times on ice at the following settings: 30 sec; power output 3; 70% duty cycle. Cell debris was removed by centrifuging at 13,000 rpm for 15 minutes at 4ºC and taking the supernatant. Supernatants were filtered through Millipore Ultrafree-MC 0.45 ?m filter units (cat. UFC30HV0S) at 13,000 rpm, 4°C for 1 minute to remove lipids. Protein concentration was determined, and extracts were aliquoted and stored at -80ºC. For a detailed protocol see http://www.modencode.org/.
Worm_chromatin_immunoprecipitation_vSS4. 0.3-3 mg extract + 1% sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl). Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 ?l of IgG dynabeads (Dyna), and washed 5 minutes with 1.5 mL of each of the following solutions: ChIP Buffer, ChIP Buffer + 1 M NaCl, ChIP Buffer + 500 mM NaCl, TEL Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and 2X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted twice with 150 ?L elution buffer for 15 minutes at 65ºC. Samples were treated with RNAse for 30 minutes at 37ºC. Samples were treated with proteinase K at 55ºC for 1-2 hrs then transfer to 65ºC overnight to reverse crosslinks. DNA was cleaned up using Zymo kit. For a detailed protocol see http://www.modencode.org/.
DNA for Library Prep is incubated with an End Repair Enzyme mix (NEB Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends. It is then purified and incubated with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends (a single A-overhang for more efficient and directed ligation of the adaptors). After a second purification, the DNA fragments is ligated with single-end ?Homebrew? adaptors which contain an index sequence within (Corbin Jones? lab). After ligation, the samples are purified TWICE using SPRI beads, allowing for a size selection step getting rid of excess adaptors. Samples are then amplified by PCR with single end primers. Depending on the original fragmentation of the DNA, samples can either be purified by SPRI or by separation and purification from an agarose gel.
Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.

value: Early Embryo

value: wild type

tag: sex type

value: mixed Male and Hermaphrodite


title: seq-UP07442_H3K9me3_DAM1411287_N2_Eemb_Input_Rep2

description: input DNA


title: seq-HK00012_H3K36me2_2C3_N2_Eemb_ChIP_Rep2

description: ChIP DNA; Antibody information listed below: official name: HK00012 H3K36me2:2C3;target name: H3K36me2;host: Mouse;antigen: KSAPATGGV(me2-K)KPHRYRPGTC;clonal: Monoclonal;purified: Protein A/G;company: Other;catalog: HK00012;short description: A protein G purified mouse monoclonal antibody to H3K36me2 (clone 2C3) obtained from Hiroshi Kimura. Used for ChIP.

value: mixed Male and Hermaphrodite population


title: Notch-GFP_W3L_ChIP-seq_ChIP_Rep1

data_processing: Solexa_Alignment protocol. Raw image files were processed following the following Illumina-compatible pipeline:1.  The Raw images were first processed with the Casava 1.7.0.2.  Base-called sequences with confidence metrics were obtained using OLB-1.9.0.  All multiplexed samples were demultiplexed using Casava 1.7.0.3. Finally the BWA program was used to align the sequence reads to the Drosophila melanogaster (v5.32) genome, allowing up to 2 edit distance in the seed region and 3 in the full read length. Tags that aligned to more than one  location were excluded from our analysis for both ChIP sample and the corresponding control sample (input chromatin). Only properly aligned reads with mapping quality more than 30 were kept. MACS_Peak-calling protocol. Peak calls were determined by using Model-based Analysis of ChIP-Seq (MACS) software, version 2 (MACS2). MACS2 was supplied with the following parameters:tagSize = 50mfold = 2genomeSize = 120000000bandwidth = 100pvalue = 1e-5  Processed data are obtained using following parameters: genome version is flybase BDGPv5

organism: Drosophila melanogaster

tag: transgene


title: seq-SN147_H4K20me1_N2_L3_ChIP_Rep2

description: ChIP DNA; Antibody information listed below: official name: DISN147 H4K20me1:001;target name: H4K20me1;host: Mouse;antigen: H4K20me1;clonal: Monoclonal;purified: Crude;company: Other;catalog: SN-147;short description: A mouse monoclonal antibody to H4K20me1 obtained from Diagenode (Cat# SN-147 lot #001). Used for ChIP.

data_processing:
ChIP-seq_alignment-BWA:JL:1 protocol. BWA is a fast light-weighted tool that aligns relatively short sequences to a sequence database. The algorithms is based on Burrows-Wheeler Transform (BWT). This protocal, was ran as DEFAULT for both bwa aln and bwa samse.bwa aln: Find the SA coordinates of the input reads. Maximum maxSeedDiff differences are allowed in the first seedLen subsequence and maximum maxDiff differences are allowed in the whole sequence.OPTIONS:-n NUM
Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]-o INT
Maximum number of gap opens [1]-e INT
Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]-d INT
Disallow a long deletion within INT bp towards the 3?-end [16]-i INT
Disallow an indel within INT bp towards the ends [5]-l INT
Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ?-k 2?. [inf]-k INT
Maximum edit distance in the seed [2]-t INT
Number of threads (multi-threading mode) [1]-M INT
Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]-O INT
Gap open penalty [11]-E INT
Gap extension penalty [4]-R INT
Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).-c
Reverse query but not complement it, which is required for alignment in the color space.-N
Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.-q INT
Parameter for read trimming. BWA trims a read down to argmax_x{\sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l is the original read length. [0] bwa samse:Generate alignments in the SAM format given single-end reads. Repetitive hits will be randomly chosen.OPTIONS:-n INT
Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]  Processed data are obtained using following parameters: genome version is ce6

growth_protocol: Worm_L3_growth_and_harvest_vPK1. About 2-7 million of worms are bleached and then hatched in M9 for 24-42 hrs. About 100 embryos are seeded onto the plate to test for contamination and hatching efficiency. Remaining hatched L1 larvae are inoculated in a proper volume of liquid culture. Next day when larvae reach the L3 stage they are cleaned by M9 washes and sucrose gradient and collected by freezing in liquid nitrogen. Just before collection DIC pictures are taken and about 50ul of worms are stained for DAPI to assess the stage.

extract_protocol:
Worm_L3_extraction_vPK1. Worms are frozen, ground, and crosslinked for 10 minutes in 1% formaldehyde. Formaldehyde is quenched and cross-linked tissue washed, then resuspended in FA buffer and subjected to sonication in Bioruptor (14 pulses of 30 seconds with 1 minute rests in between). Extracts are then spun down and soluble fraction is stored for quality tests and future ChIP.
Worm_chromatin_immunoprecipitation_vIL2. Appropriate amount of extract is incubated overnight with a proper amount of antibody (exceptional antibodies due to better results are incubated 2hrs). Afterwards, 40ul of equilibrated magnetic beads (either protein A or G, depending on antibody) are added and incubated for 2 hrs. Later, washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer are performed and DNA is eluted in elution buffer (1% SDS in TE with 250 mM NaCl) ? two times with 57 ?l volume each, at 65°C. Samples are treated with RNAse, proteinase K and then crosslinks are reversed overnight at 65°C. DNA is purified on Qiagen PCR purification columns, tested by q-PCR for ChIP quality, and stored at -20°C for future applications.
ChIP or input DNA is blunt ended, A-tailed, ligated to adaptors, amplified by PCR, then size selected using AMPure beads.  The protocol uses the Illuimina TruSeq DNA Sample Prep Kit.
Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.

value: L3 Larva


title: yki_8-16h_embryonic_ChIP-seq_Input_Rep1

description: input DNA

extract_protocol:
1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder.   2. For a 8-16 hours egg laying, new plates are added after a 2 hours pre-egglaying at 12 pm and flies are allowed to lay eggs for 8 hours.   3. Embryos are collected at 7.45 am using a filter mesh and a brush. They are rinsed with EWB and cross-linked at 8am in 1.8% formaldehyde at room temperature for 15 min.
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911).  After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.

value: Y cn bw sp

value: Embryo 8-16h


title: seq-SN147_H4K20me1_N2_L3_ChIP_Rep1


title: Notch-GFP_W3L_ChIP-seq_Input_Rep2

description: input DNA


title: seq-SN147_H4K20me1_N2_L3_Input_Rep2

description: input DNA


title: Notch-GFP_W3L_ChIP-seq_Input_Rep1

description: input DNA


title: lola-PI-GFP_W3L_ChIP-seq_ChIP_Rep2

value: lola-RI-GFP

value: lola-RI genomic coding region


title: lola-PI-GFP_W3L_ChIP-seq_ChIP_Rep1


title: seq-SN147_H4K20me1_N2_L3_Input_Rep1

description: input DNA


title: insv_Kc_cells_ChIP-seq_ChIP_Rep3

description: ChIP DNA; Antibody information listed below: official name: insv;target name: INSV;host: Rabbit;antigen: MENQYQRIVSATAREDSQARRSKPLFLNAWSQTSSVDFEVLRSISAPDPQ VEVENRALRDKVRYLEAKLQQHKDLLSQIHATSARMQQASSLLAESRPPT PPAVQSHHILTPPSSEICAPAQNPQILDYKIISAPDDADAIEIRLAAESL NSLSTSADSDRLEICLGDENHQQSNHHNSQQQYRISNGIKRDGSSESADT PLQFIKRRKLQEIQLQEEIPRQNIKLPAKPQVKARNGSFTDLNKGQQQNM DNVMVSIGPNNTCVPASVFENINWSVCSLATRKLLVTIFDRETLATHSMT GKPSPAFKDQDKPLKRMLDPGKIQDIIFAVTHKCNASEKEVRNAITTKCA DENKMMKIQNVKRRSSGIKHEKENII;clonal: Polyclonal;purified: Size;company: Lab;catalog: Eric Lai Lab;short description: This is a gift from Eric Lai to Nicolas Negre for the modENCODE project. This antibody has been pre-adsorbed with 0-1h old Drosophila embryos before use in ChIP.

extract_protocol:
Cell lines (S2, Kc167...) are grown as described by the DGRC and the Cherbas Lab. After expanding the cell line, cells are transfered in a plate. Once confluent, 1.8% formaldehyde is added to the cell culture. Cells are harvested in presence of the formaldehyde with the help of a cell scrapper. After 15 minutes of incubation at room temperature, the cross-linking reaction is quenched with glycine for 5 minutes. Cells pellet are then washed 3 times with lysis buffer. Regular chromatin extraction before sonication is then performed.
Formaldehyde Cross-linking of Chromatin from any Drosophila tissue (Embryos, Larvae, Pupae, Adult flies).1. Homogenize the material (about 150-200 mg of embryos, pupae, adµlts, larvae, sufficient for four-five independent immuno-precipitations) in 5 ml of buffer A1 + 1.8% formaldehyde (290 µl of 37% solution) at room temperature using first a Potter homogenizer and then a Douncer with type A pestle (3 strokes). Wait 15? (total time starting from beginning of homogenization). Add glycin solution to 225 mM (540 µl of 2.5 M solution for 6 ml of cross-linked mixture), mix and incubate 5?. Put on ice. Keep on ice unless specified.2. Transfer the homogenate to a 15 ml tube. Centrifuge 5 min, at 4000 g at 4°C. Discard supernatant. Add 3 ml of buffer A1, resuspend pellet and spin down the same way. Repeat washing step 3 times.3. Wash once in 3 ml of lysis buffer without SDS. Spin down, 5 min at 4000g.4. Resuspend cross-linked material in 0.5 ml of lysis buffer and add SDS to 0.1% (2.5 µl) and N-lauroylsarcosine to 0.5% (12.5 µl). Incubate 10 min at 4°C in a rotating wheel.5. Sonicate in Polypropylene 15ml Falcon tubes according to .Diagenode instructions. The settings are: High power, 15 minutes, 30 seconds off, 30 seconds on. The water bath shoµld be kept cold either in a cold room or by a cold water pump.6. Rotate 10'. Transfer to eppendorf tubes and centrifuge 5', room temperature at maximum speed. Transfer supernatant to the new tube. Add another 0.5 ml of lysis buffer to the pellet, rotate for 10 min. Repeat centrifugation and combine supernatants. Centrifuge the combined supernatant 2x10 min at max speed. The supernatant from this stage will be referred as chromatin extract. Chromatin can be stored at ?80°C for several months at this stage. Add sodium azide to 0.02% for storage.7. [Optional : Purification of chromatin. Put chromatin extract in Centricon YM-100 column (4212), centrifuge 3x40' (or more) at 1000 g while adding lysis buffer. At least 3 vol. of lysis buffer should pass through column. Bring the final volume of chromatin extract to 1 ml by lysis buffer + protease inhibitors.]
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911).  After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.

tag: cell line

tag: tissue

value: embryo-derived cell-line

value: late embryonic stage

value: se/e

value: Female


title: lola-PI-GFP_W3L_ChIP-seq_Input_Rep1

description: input DNA

value: Unknown


title: lola-PI-GFP_W3L_ChIP-seq_Input_Rep2

description: input DNA


title: seq-ab1791_H3_377098_N2_Eemb_ChIP_Rep1

description: ChIP DNA; Antibody information listed below: official name: C. elegans  AB1791 H3 rabbit polyclonal antibody;target name: Histone H3;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 100 to the C-terminus of Human Histone H3;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: AB1791;short description: An affinity purified rabbit polyclonal antibody to H3 obtained from Abcam (H3-AB1791);used for ChIP.

data_processing:
ChIP-seq_alignment-BWA:JL:1 protocol. BWA is a fast light-weighted tool that aligns relatively short sequences to a sequence database. The algorithms is based on Burrows-Wheeler Transform (BWT). This protocal, was ran as DEFAULT for both bwa aln and bwa samse.bwa aln: Find the SA coordinates of the input reads. Maximum maxSeedDiff differences are allowed in the first seedLen subsequence and maximum maxDiff differences are allowed in the whole sequence.OPTIONS:-n NUM
Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]-o INT
Maximum number of gap opens [1]-e INT
Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]-d INT
Disallow a long deletion within INT bp towards the 3?-end [16]-i INT
Disallow an indel within INT bp towards the ends [5]-l INT
Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ?-k 2?. [inf]-k INT
Maximum edit distance in the seed [2]-t INT
Number of threads (multi-threading mode) [1]-M INT
Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]-O INT
Gap open penalty [11]-E INT
Gap extension penalty [4]-R INT
Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).-c
Reverse query but not complement it, which is required for alignment in the color space.-N
Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.-q INT
Parameter for read trimming. BWA trims a read down to argmax_x{\sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l is the original read length. [0] bwa samse:Generate alignments in the SAM format given single-end reads. Repetitive hits will be randomly chosen.OPTIONS:-n INT
Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]  Processed data are obtained using following parameters: genome version is ce6   BEADS_Normalization protocol. Bias Elimination Algorithm for Deep Sequencing (BEADS): Sequence biasesin the Input and ChIP samples were normalized to remove: GC contentbiases, mappability biases, and regional biases. The originalPython/Java implementation can be accessed athttp://beads.sourceforge.net. Histone_Replicates_QC protocol. After BEADS normalization, genome wide correlation of BEADS signal was calculated in 1 kb windows, with a requirement for replicate correlation of at least 0.7. Signal tracks of biological replicates were also inspected visually for reproducibility.

growth_protocol: Worm_embryo_growth_and_harvest_vSS4. Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Live embryos were cross-linked in M9 + 1.85% formaldehyde for 30 minutes at room temperature. Embryos were then washed twice with M9 Buffer, once with 100 mM Tris-HCl pH 7.5 and once with FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl). Pellets were frozen at -80C. For a detailed protocol see http://www.modencode.org/.

extract_protocol:
Worm_embryo_extraction_vSS2. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl) + protease inhibitors. Sample was dounced 30 times using tight pestle at 4ºC, then sonicated 9 times on ice at the following settings: 30 sec; power output 3; 70% duty cycle. Cell debris was removed by centrifuging at 13,000 rpm for 15 minutes at 4ºC and taking the supernatant. Supernatants were filtered through Millipore Ultrafree-MC 0.45 ?m filter units (cat. UFC30HV0S) at 13,000 rpm, 4°C for 1 minute to remove lipids. Protein concentration was determined, and extracts were aliquoted and stored at -80ºC. For a detailed protocol see http://www.modencode.org/.
Worm_chromatin_immunoprecipitation_vIL2. Appropriate amount of extract is incubated overnight with a proper amount of antibody (exceptional antibodies due to better results are incubated 2hrs). Afterwards, 40ul of equilibrated magnetic beads (either protein A or G, depending on antibody) are added and incubated for 2 hrs. Later, washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer are performed and DNA is eluted in elution buffer (1% SDS in TE with 250 mM NaCl) ? two times with 57 ?l volume each, at 65°C. Samples are treated with RNAse, proteinase K and then crosslinks are reversed overnight at 65°C. DNA is purified on Qiagen PCR purification columns, tested by q-PCR for ChIP quality, and stored at -20°C for future applications.
ChIP or input DNA is blunt ended, A-tailed, ligated to adaptors, amplified by PCR, then size selected using AMPure beads.  The protocol uses the Illuimina TruSeq DNA Sample Prep Kit.
Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.


title: seq-ab1791_H3_377098_N2_Eemb_ChIP_Rep2


title: seq-ab1791_H3_377098_N2_Eemb_Input_Rep2

description: input DNA


title: seq-ab1791_H3_377098_N2_Eemb_Input_Rep1

description: input DNA


title: insv_Kc_cells_ChIP-seq_ChIP_Rep1


title: insv_Kc_cells_ChIP-seq_Input_Rep3

description: input DNA


title: insv_Kc_cells_ChIP-seq_Input_Rep1

description: input DNA


title: seq-SDQ0835_SCC1_FEM2_AD_ChIP_Rep1

description: ChIP DNA; Antibody information listed below: official name: SCC-1 SDQ0835;target name: SCC-1;host: Rabbit;antigen: TPSMEAQAEPWEDLNLNEDIQEDIQAQGPAVDEFFNDVRMDDDDDRQPAQEMDFGDNFDFPQEVEHQECAPIPIQSGFAGENKENEDAEDWSDPFGSSNS;clonal: Polyclonal;purified: Affinity;company: SDI;catalog: SDQ0835;short description: An Affinity purified Rabbit Polyclonal antibody to SCC-1 obtained from SDI (SCC-1 SDQ0835)

data_processing:
ChIP-seq_alignment-BWA:JL:1 protocol. BWA is a fast light-weighted tool that aligns relatively short sequences to a sequence database. The algorithms is based on Burrows-Wheeler Transform (BWT). This protocal, was ran as DEFAULT for both bwa aln and bwa samse.bwa aln: Find the SA coordinates of the input reads. Maximum maxSeedDiff differences are allowed in the first seedLen subsequence and maximum maxDiff differences are allowed in the whole sequence.OPTIONS:-n NUM
Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]-o INT
Maximum number of gap opens [1]-e INT
Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]-d INT
Disallow a long deletion within INT bp towards the 3?-end [16]-i INT
Disallow an indel within INT bp towards the ends [5]-l INT
Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ?-k 2?. [inf]-k INT
Maximum edit distance in the seed [2]-t INT
Number of threads (multi-threading mode) [1]-M INT
Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]-O INT
Gap open penalty [11]-E INT
Gap extension penalty [4]-R INT
Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).-c
Reverse query but not complement it, which is required for alignment in the color space.-N
Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.-q INT
Parameter for read trimming. BWA trims a read down to argmax_x{\sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l is the original read length. [0] bwa samse:Generate alignments in the SAM format given single-end reads. Repetitive hits will be randomly chosen.OPTIONS:-n INT
Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]  Processed data are obtained using following parameters: genome version is ce10   ChIP-Seq_MACS_Peak-calling_over_reps:JL:1 protocol. Peak calls were determined by using Model-based Analysis of ChIP-Seq (MACS) software. MACS was supplied with the following parameters:tagSize = 25mfold = 10,30genomeSize = 90000000bandwidth = 300pvalue = 1e-5  Processed data are obtained using following parameters: genome version is ce10


title: insv_Kc_cells_ChIP-seq_Input_Rep2

description: input DNA


title: Hr78-GFP_8-16_embryonic_ChIP-seq_ChIP_Rep1

extract_protocol:
1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder.2. For a 8-16 hours egg laying, new plates are added after a 2 hours pre-egglaying at 12 pm and flies are allowed to lay eggs for 8 hours.3. Embryos are collected at 7.45 am using a filter mesh and a brush. They are rinsed with EWB and cross-linked at 8am in 1.8% formaldehyde at room temperature for 15 min.
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911).  After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.

value: Hr78 genomic coding region


title: Hr78-GFP_8-16_embryonic_ChIP-seq_ChIP_Rep2


title: Hr78-GFP_8-16_embryonic_ChIP-seq_Input_Rep2

description: input DNA


title: seq-SDQ2370_LIN53_N2_LTemb_ChIP_Rep2

description: ChIP DNA; Antibody information listed below: official name: SDQ2370-LIN53;target name: LIN-53;host: Rabbit;antigen: SLSVQWLPDVAKDNSDHTIHRLILGTHTSDEQNHLLISKICMPTDDAQFDASRYDTERSEYGGFGAVNGKVEPDIRINHEGEVNRARYMPQKSNIIATKS;clonal: Polyclonal;purified: Affinity;company: SDI;catalog: G3048-048A02;short description: An affinity purified rabbit polyclonal antibody to LIN-53 obtained from SDI (LIN3-Q2370)

data_processing:
Illumina_DNA_Sequencing:JL:1 protocol. Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.  Processed data are obtained using following parameters: read length is 36   ChIP-seq_alignment-BWA:JL:1 protocol. BWA is a fast light-weighted tool that aligns relatively short sequences to a sequence database. The algorithms is based on Burrows-Wheeler Transform (BWT). This protocal, was ran as DEFAULT for both bwa aln and bwa samse.bwa aln: Find the SA coordinates of the input reads. Maximum maxSeedDiff differences are allowed in the first seedLen subsequence and maximum maxDiff differences are allowed in the whole sequence.OPTIONS:-n NUM
Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]-o INT
Maximum number of gap opens [1]-e INT
Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]-d INT
Disallow a long deletion within INT bp towards the 3?-end [16]-i INT
Disallow an indel within INT bp towards the ends [5]-l INT
Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ?-k 2?. [inf]-k INT
Maximum edit distance in the seed [2]-t INT
Number of threads (multi-threading mode) [1]-M INT
Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]-O INT
Gap open penalty [11]-E INT
Gap extension penalty [4]-R INT
Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).-c
Reverse query but not complement it, which is required for alignment in the color space.-N
Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.-q INT
Parameter for read trimming. BWA trims a read down to argmax_x{\sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l is the original read length. [0] bwa samse:Generate alignments in the SAM format given single-end reads. Repetitive hits will be randomly chosen.OPTIONS:-n INT
Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]  Processed data are obtained using following parameters: genome version is WS220   ChIP-Seq_MACS_Peak-calling_over_reps:JL:1 protocol. Peak calls were determined by using Model-based Analysis of ChIP-Seq (MACS) software. MACS was supplied with the following parameters:tagSize = 25mfold = 10,30genomeSize = 90000000bandwidth = 300pvalue = 1e-5  Processed data are obtained using following parameters: genome version is WS220

growth_protocol: Worm_embryo_growth_and_harvest_vSS7. Embryos were prepared by bleaching from gravid N2 adults grown in standard S-basal media liquid culture. Embryos were incubated for 3.5 hours in M9 then frozen in liquid nitrogen.

extract_protocol:
Worms are frozen, ground, and crosslinked for 10 minutes in 1% formaldehyde. Later,washed pellets are resuspended in FA buffer and subjected to sonication in a DiagenodeBioruptor (40 pulses of 30 seconds with 1 minute rests in between at full power). Extractsare then spun down and the soluble fraction is stored for quality tests and future ChIP.
Worm_chromatin_immunoprecipitation_vSS4. 0.3-3 mg extract + 1% sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl). Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 ?l of IgG dynabeads (Dyna), and washed 5 minutes with 1.5 mL of each of the following solutions: ChIP Buffer, ChIP Buffer + 1 M NaCl, ChIP Buffer + 500 mM NaCl, TEL Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and 2X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted twice with 150 ?L elution buffer for 15 minutes at 65ºC. Samples were treated with RNAse for 30 minutes at 37ºC. Samples were treated with proteinase K at 55ºC for 1-2 hrs then transfer to 65ºC overnight to reverse crosslinks. DNA was cleaned up using Zymo kit. For a detailed protocol see http://www.modencode.org/.
Short description for UNC NimbleGen Database:Both ChIP and input DNA samples have their ends blunted and phosphorylated with KlenowDNAP, T4 DNAP, and T4 PNK. 3?-dA overhangs are then added with Klenow Fragment (3? to 5?exo minus), and Illumina adapters are subsequently ligated to the ends. Next, PCRamplification is performed using Illumina 1.1 and 2.1 primers, for a total of 18 cycles. AmplifiedDNA libraries are size-selected on a 2% agarose gel so that fragments sized between 250-350bp are obtained; gel extraction is carried out at room temperature.

value: Late Embryos


title: Hr78-GFP_8-16_embryonic_ChIP-seq_Input_Rep1

description: input DNA


title: seq-SDQ0811_RAD51_FEM2_AD_Input_Rep1

description: input DNA

extract_protocol:
Worm extract was made from adult worms.  Frozen worm popcorn was ground up in a mixer mill.  Samples were then fixed with 1% formaldehyde for 10 minutes. Chromatin was then sheared by sonication in the Bioruptor, and the soluble fraction was collected and stored at -80°C for future use.
Worm extract from adult worms and 5 ?g of affinity purified antibody was used for ChIP. Dynal Protein A or G beads were used to recover the ChIPed DNA.  Samples were then treated with RNase A and Proteinase K, and reverse crosslinked at 65°C. ChIPed DNA was purified and then quantified using the Pico Green HS fluorescent assay.
DNA for Library Prep is incubated with an End Repair Enzyme mix (NEB Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends. It is then purified and incubated with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends (a single A-overhang for more efficient and directed ligation of the adaptors). After a second purification, the DNA fragments is ligated with single-end ?Homebrew? adaptors which contain an index sequence within (Corbin Jones? lab). After ligation, the samples are purified TWICE using SPRI beads, allowing for a size selection step getting rid of excess adaptors. Samples are then amplified by PCR with single end primers. Depending on the original fragmentation of the DNA, samples can either be purified by SPRI or by separation and purification from an agarose gel.
Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.

value: Germline containing young adult

value: Hermaphrodite


title: H3K4me3_S2_cells_ChIP-seq_ChIP_Rep2

description: ChIP DNA; Antibody information listed below: official name: H3K4me3;target name: H3K4me3;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3 tri methylated at K4.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab8580;short description: Histone H3 (tri methyl K4) antibody - ChIP Grade

value: Male


title: seq-SDQ0801_HIM17_FEM2_AD_ChIP_Rep2

description: ChIP DNA; Antibody information listed below: official name: HIM-17 SDQ0801;target name: HIM-17;host: Rabbit;antigen: RKLDDDESNDVGAYFEDDNQQGTSYRTPFMPSHLGLIQDQSPDLTMKDLFNRKRSRPQSTGFIDHSQARYYDRYKQQEYTRRNDFMKYCHVCRKKTVGKL;clonal: Polyclonal;purified: Affinity;company: SDI;catalog: GA2945;short description: An affinity purified rabbit polyclonal antibody to HIM-17 obtained from SDI (HIM17-Q0801)

extract_protocol:
Worm extract was made from adult worms.  Frozen worm popcorn was ground up in a mixer mill.  Samples were then fixed with 1% formaldehyde for 10 minutes. Chromatin was then sheared by sonication in the Bioruptor, and the soluble fraction was collected and stored at -80°C for future use.
Worm extract from adult worms and 5 ?g of affinity purified antibody was used for ChIP. Dynal Protein A or G beads were used to recover the ChIPed DNA.  Samples were then treated with RNase A and Proteinase K, and reverse crosslinked at 65°C. ChIPed DNA was purified and then quantified using the Pico Green HS fluorescent assay.
Libraries were prepared according to Illumina's instructions for ?Preparing Samples for ChIP Sequencing of DNA?, with some variation. DNA was end-repaired using T4 DNA polymerase, Klenow DNA polymerase and T4 DNA polymerase.  A?s were then added to the 3? ends of the fragments using Kelow fragment (3? ? 5? exo minus). Paired-end adapters were then ligated onto the ends of the fragments.  DNA library was size selected by gel purification, fragments of 250-500 bp were band isolated from an agarose gel. DNA was PCR amplified for 18 cycles. The Bioanalyzer was used to verify fragment purification and quantify DNA.
Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.


title: seq-SDQ0801_HIM17_FEM2_AD_ChIP_Rep1


title: H3K4me3_S2_cells_ChIP-seq_Input_Rep2

description: input DNA


title: H3K4me3_8-16h_embryonic_ChIP-seq_ChIP_Rep2


title: H3K4me3_S2_cells_ChIP-seq_ChIP_Rep1


title: H3K4me3_S2_cells_ChIP-seq_Input_Rep1

description: input DNA


title: seq-SDQ0801_HIM17_FEM2_AD_Input_Rep1

description: input DNA


title: H3K4me3_8-16h_embryonic_ChIP-seq_Input_Rep2

description: input DNA


title: seq-SDQ2370_LIN53_N2_L3_ChIP_Rep2

data_processing:
ChIP-seq_alignment-BWA:JL:1 protocol. BWA is a fast light-weighted tool that aligns relatively short sequences to a sequence database. The algorithms is based on Burrows-Wheeler Transform (BWT). This protocal, was ran as DEFAULT for both bwa aln and bwa samse.bwa aln: Find the SA coordinates of the input reads. Maximum maxSeedDiff differences are allowed in the first seedLen subsequence and maximum maxDiff differences are allowed in the whole sequence.OPTIONS:-n NUM
Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]-o INT
Maximum number of gap opens [1]-e INT
Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]-d INT
Disallow a long deletion within INT bp towards the 3?-end [16]-i INT
Disallow an indel within INT bp towards the ends [5]-l INT
Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ?-k 2?. [inf]-k INT
Maximum edit distance in the seed [2]-t INT
Number of threads (multi-threading mode) [1]-M INT
Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]-O INT
Gap open penalty [11]-E INT
Gap extension penalty [4]-R INT
Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).-c
Reverse query but not complement it, which is required for alignment in the color space.-N
Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.-q INT
Parameter for read trimming. BWA trims a read down to argmax_x{\sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l is the original read length. [0] bwa samse:Generate alignments in the SAM format given single-end reads. Repetitive hits will be randomly chosen.OPTIONS:-n INT
Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]  Processed data are obtained using following parameters: genome version is ce6 (both wiggle and peak calls)   ChIP-Seq_MACS_Peak-calling:JL:1 protocol. Peak calls were determined by using Model-based Analysis of ChIP-Seq (MACS) software. MACS was supplied with the following parameters:tagSize = 25mfold = 10,30genomeSize = 90000000bandwidth = 300pvalue = 1e-5  Processed data are obtained using following parameters: genome version is ce6 (both wiggle and peak calls)

extract_protocol:
Worm_L3_extraction_vPK1. Worms are frozen, ground, and crosslinked for 10 minutes in 1% formaldehyde. Formaldehyde is quenched and cross-linked tissue washed, then resuspended in FA buffer and subjected to sonication in Bioruptor (14 pulses of 30 seconds with 1 minute rests in between). Extracts are then spun down and soluble fraction is stored for quality tests and future ChIP.
Worm_chromatin_immunoprecipitation_vIL2. Appropriate amount of extract is incubated overnight with a proper amount of antibody (exceptional antibodies due to better results are incubated 2hrs). Afterwards, 40ul of equilibrated magnetic beads (either protein A or G, depending on antibody) are added and incubated for 2 hrs. Later, washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer are performed and DNA is eluted in elution buffer (1% SDS in TE with 250 mM NaCl) ? two times with 57 ?l volume each, at 65°C. Samples are treated with RNAse, proteinase K and then crosslinks are reversed overnight at 65°C. DNA is purified on Qiagen PCR purification columns, tested by q-PCR for ChIP quality, and stored at -20°C for future applications.
DNA was incubated with an enzyme mix (Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends and then with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends. The DNA fragments were ligated with appropriate adaptor (Illumina) and then amplified by PCR. The amplicon was loaded into an agarose gel, and size-selected DNA was recovered from the gel.
Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.

value: wild type

value: mixed Male and Hermaphrodite


title: seq-SDQ0801_HIM17_FEM2_AD_Input_Rep2

description: input DNA


title: seq-SDQ2370_LIN53_N2_L3_Input_Rep2

description: input DNA

value: L3 Larva


title: seq-SDQ2370_LIN53_N2_L3_ChIP_Rep1

data_processing:
ChIP-seq_alignment-BWA:JL:1 protocol. BWA is a fast light-weighted tool that aligns relatively short sequences to a sequence database. The algorithms is based on Burrows-Wheeler Transform (BWT). This protocal, was ran as DEFAULT for both bwa aln and bwa samse.bwa aln: Find the SA coordinates of the input reads. Maximum maxSeedDiff differences are allowed in the first seedLen subsequence and maximum maxDiff differences are allowed in the whole sequence.OPTIONS:-n NUM
Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]-o INT
Maximum number of gap opens [1]-e INT
Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]-d INT
Disallow a long deletion within INT bp towards the 3?-end [16]-i INT
Disallow an indel within INT bp towards the ends [5]-l INT
Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ?-k 2?. [inf]-k INT
Maximum edit distance in the seed [2]-t INT
Number of threads (multi-threading mode) [1]-M INT
Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]-O INT
Gap open penalty [11]-E INT
Gap extension penalty [4]-R INT
Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).-c
Reverse query but not complement it, which is required for alignment in the color space.-N
Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.-q INT
Parameter for read trimming. BWA trims a read down to argmax_x{\sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l is the original read length. [0] bwa samse:Generate alignments in the SAM format given single-end reads. Repetitive hits will be randomly chosen.OPTIONS:-n INT
Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]  Processed data are obtained using following parameters: genome version is ce6 (both wiggle and peak calls)   ChIP-Seq_MACS_Peak-calling_over_reps:JL:1 protocol. Peak calls were determined by using Model-based Analysis of ChIP-Seq (MACS) software. MACS was supplied with the following parameters:tagSize = 25mfold = 10,30genomeSize = 90000000bandwidth = 300pvalue = 1e-5  Processed data are obtained using following parameters: genome version is ce6 (both wiggle and peak calls)


title: seq-SDQ2370_LIN53_N2_LTemb_ChIP_Rep1


title: seq-SDQ2370_LIN53_N2_LTemb_Input_Rep2

description: input DNA

data_processing:
Illumina_DNA_Sequencing:JL:1 protocol. Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.  Processed data are obtained using following parameters: read length is 36   ChIP-seq_alignment-BWA:JL:1 protocol. BWA is a fast light-weighted tool that aligns relatively short sequences to a sequence database. The algorithms is based on Burrows-Wheeler Transform (BWT). This protocal, was ran as DEFAULT for both bwa aln and bwa samse.bwa aln: Find the SA coordinates of the input reads. Maximum maxSeedDiff differences are allowed in the first seedLen subsequence and maximum maxDiff differences are allowed in the whole sequence.OPTIONS:-n NUM
Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]-o INT
Maximum number of gap opens [1]-e INT
Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]-d INT
Disallow a long deletion within INT bp towards the 3?-end [16]-i INT
Disallow an indel within INT bp towards the ends [5]-l INT
Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ?-k 2?. [inf]-k INT
Maximum edit distance in the seed [2]-t INT
Number of threads (multi-threading mode) [1]-M INT
Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]-O INT
Gap open penalty [11]-E INT
Gap extension penalty [4]-R INT
Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).-c
Reverse query but not complement it, which is required for alignment in the color space.-N
Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.-q INT
Parameter for read trimming. BWA trims a read down to argmax_x{\sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l is the original read length. [0] bwa samse:Generate alignments in the SAM format given single-end reads. Repetitive hits will be randomly chosen.OPTIONS:-n INT
Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]  Processed data are obtained using following parameters: genome version is WS220   ChIP-Seq_MACS_Peak-calling:JL:1 protocol. Peak calls were determined by using Model-based Analysis of ChIP-Seq (MACS) software. MACS was supplied with the following parameters:tagSize = 25mfold = 10,30genomeSize = 90000000bandwidth = 300pvalue = 1e-5  Processed data are obtained using following parameters: genome version is WS220


title: seq-SDQ2370_LIN53_N2_LTemb_Input_Rep1

description: input DNA


title: seq-SDQ5421_CEC7_fem2_AD_ChIP_Rep1

description: ChIP DNA; Antibody information listed below: official name: Y37D8A.11 SDQ5421;target name: CEC-7;host: Rabbit;antigen: EAPKETVQKKARIEPVDSAEESKAKGSTAPMRKSAKKVGESNSEATDPEAKAKESSQSALKVEPPLTTHNSSRKTVEATGAMKRLTVAKETEDEALKSQKKARIEPVEDASKAKGSAAPKSASSRAEDGESSSTNDAAGTR;clonal: Polyclonal;purified: Affinity;company: SDI;catalog: SDQ5421;short description: An Affinity purified Rabbit Polyclonal antibody to CEC-7 obtained from SDI (Y37D8A.11 SDQ5421)

extract_protocol:
Worm extract was made from adult worms.  Frozen worm popcorn was ground up in a mixer mill.  Samples were then fixed with 1% formaldehyde for 10 minutes. Chromatin was then sheared by sonication in the Bioruptor, and the soluble fraction was collected and stored at -80°C for future use.
Worm_chromatin_immunoprecipitation_vCW2. ~100ug (~2ug DNA) extract was used for each ChIP with 5% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl) and treated with RNAse. Antibody was added to each IP sample and incubated overnight at 4C. Immune complexes were incubated (2hrs at 4C) with 50 uL protein A or anti-IgG Dynabeads and washed 5 minutes with 1mL of each of the following solutions: ChIP Buffer, ChIP Buffer+1M NaCl, ChIP Buffer+500mM NaCl, TEL Buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA), and 2X TE (10mM Tris-HCl pH 8.0, 1mM EDTA). Samples were eluted twice with 75uL elution buffer for 15 minutes at 65C. Samples were treated with proteinase K at 55C for 1-2hrs then transferred to 65C overnight to reverse crosslinks. DNA was cleaned up with Ampure XP beads. For a detailed protocol see http://www.modencode.org/.
Worm_ChIP-seq_ DNA_Library_Preparation_vSS2. ChIP and 10ng of input are blunted (T4 polymerase), an A-overhang is added (Klenow), DNA is ligated (concentrated T4 ligase) to annealed adaptors, size-selected, and amplified by PCR.Changes: Ligate O/N at 16C. Use adapters and primer cocktail from Illumina TruSeq kit. Use AMPure cleanup in the place of Qiagne cleanup. Use Pippin prep for size selection .
Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.


title: seq-SDQ5421_CEC7_fem2_AD_Input_Rep1

description: input DNA


title: H3K4me3_8-16h_embryonic_ChIP-seq_Input_Rep1

description: input DNA

value: Y cn bw sp


title: H3K4me1_W3L_ChIP-seq_Input_Rep2

description: input DNA

extract_protocol:
1. y; bw cn sp flies are cultivated in standard fly food covered with yeast powder.2. Flies are removed from the bottles after 24 hours and eggs are allowed to grow at 25 C for several days until they reach the desired growth stage.3. Wandering third instal larvae are collected from the side of the bottles using a brush. They are rinsed with EWB and cross-linked in 1.8% formaldehyde at room temperature for 15 min.
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911).  After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.


title: H3K4me1_W3L_ChIP-seq_Input_Rep1

description: input DNA


title: H3K4me1_W3L_ChIP-seq_ChIP_Rep2

description: ChIP DNA; Antibody information listed below: official name: H3K4me1;target name: H3K4me1;host: Rabbit;antigen: Polyclonal rabbit antibody raised against synthetic peptide derived from residues 1-100 of H3 monomethylated at K4.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab8895;short description: Polyclonal rabbit antibody raised against synthetic peptide derived from residues 1-100 of H3 monomethylated at K4.


title: seq-ab817_8WG16_639746_N2_Eemb_ChIP_Rep2

description: ChIP DNA; Antibody information listed below: official name: ABAB817 8WG16:346588;target name: RNA polII CTD domain unphosophorylated;host: Mouse;antigen: Native RNA Polymerase II purified from wheat germ;clonal: Monoclonal;purified: Size;company: Abcam;catalog: ab817;short description: A mouse monoclonal antibody which recognizes primarily the unphosphorylated RNA Pol II CTD (8WG16) was used for ChIP.

data_processing:
Illumina_DNA_Sequencing:JL:1 protocol. Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.  Processed data are obtained using following parameters: read length is 36   ChIP-seq_alignment-BWA:JL:1 protocol. BWA is a fast light-weighted tool that aligns relatively short sequences to a sequence database. The algorithms is based on Burrows-Wheeler Transform (BWT). This protocal, was ran as DEFAULT for both bwa aln and bwa samse.bwa aln: Find the SA coordinates of the input reads. Maximum maxSeedDiff differences are allowed in the first seedLen subsequence and maximum maxDiff differences are allowed in the whole sequence.OPTIONS:-n NUM
Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]-o INT
Maximum number of gap opens [1]-e INT
Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]-d INT
Disallow a long deletion within INT bp towards the 3?-end [16]-i INT
Disallow an indel within INT bp towards the ends [5]-l INT
Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ?-k 2?. [inf]-k INT
Maximum edit distance in the seed [2]-t INT
Number of threads (multi-threading mode) [1]-M INT
Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]-O INT
Gap open penalty [11]-E INT
Gap extension penalty [4]-R INT
Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).-c
Reverse query but not complement it, which is required for alignment in the color space.-N
Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.-q INT
Parameter for read trimming. BWA trims a read down to argmax_x{\sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l is the original read length. [0] bwa samse:Generate alignments in the SAM format given single-end reads. Repetitive hits will be randomly chosen.OPTIONS:-n INT
Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]  Processed data are obtained using following parameters: genome version is ce10   ChIP-Seq_MACS_Peak-calling:JL:1 protocol. Peak calls were determined by using Model-based Analysis of ChIP-Seq (MACS) software. MACS was supplied with the following parameters:tagSize = 25mfold = 10,30genomeSize = 90000000bandwidth = 300pvalue = 1e-5  Processed data are obtained using following parameters: genome version is ce10

extract_protocol:
Worm_embryo_extraction_vSS5. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl) + protease inhibitors. Sample was dounced 30 times using tight pestle at 4ºC, then sonicated 14 times in ice water bath followed by 1 minute pause at the highest setting. Cell debris was removed by centrifuging at 13,000 rpm for 15 minutes at 4ºC and taking the supernatant. Supernatants were filtered through Millipore Ultrafree-MC 0.45 ?m filter units (cat. UFC30HV0S) at 13,000 rpm, 4°C for 1 minute to remove lipids. Protein concentration was determined, and extracts were aliquoted and stored at -80ºC. For a detailed protocol see http://www.modencode.org/.
Worm_chromatin_immunoprecipitation_vSS4. 0.3-3 mg extract + 1% sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl). Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 ?l of IgG dynabeads (Dyna), and washed 5 minutes with 1.5 mL of each of the following solutions: ChIP Buffer, ChIP Buffer + 1 M NaCl, ChIP Buffer + 500 mM NaCl, TEL Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and 2X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted twice with 150 ?L elution buffer for 15 minutes at 65ºC. Samples were treated with RNAse for 30 minutes at 37ºC. Samples were treated with proteinase K at 55ºC for 1-2 hrs then transfer to 65ºC overnight to reverse crosslinks. DNA was cleaned up using Zymo kit. For a detailed protocol see http://www.modencode.org/.
DNA for Library Prep is incubated with an End Repair Enzyme mix (NEB Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends. It is then purified and incubated with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends (a single A-overhang for more efficient and directed ligation of the adaptors). After a second purification, the DNA fragments is ligated with single-end ?Homebrew? adaptors which contain an index sequence within (Corbin Jones? lab). After ligation, the samples are purified TWICE using SPRI beads, allowing for a size selection step getting rid of excess adaptors. Samples are then amplified by PCR with single end primers. Depending on the original fragmentation of the DNA, samples can either be purified by SPRI or by separation and purification from an agarose gel.

value: Early Embryo

value: mixed Male and Hermaphrodite population


title: H3K4me1_S2_cells_ChIP-seq_ChIP_Rep2


title: seq-ab817_8WG16_639746_N2_Eemb_ChIP_Rep1


title: H3K4me1_S2_cells_ChIP-seq_ChIP_Rep1


title: H3K4me3_8-16h_embryonic_ChIP-seq_ChIP_Rep1

value: Embryo 8-16h


title: seq-SDQ5413_CEC7_fem2_AD_Input_Rep2

description: input DNA


title: seq-SDQ2370_LIN53_N2_L3_Input_Rep1

description: input DNA


title: cmi_0-8h_embryonic_ChIP-seq_ChIP_Rep2

description: ChIP DNA; Antibody information listed below: official name: anti-Cmi;target name: CG5591;host: Rabbit;antigen: full length protein;clonal: Polyclonal;purified: Affinity;company: Lab;catalog: Andrew Dingwall Lab;short description: This is a gift from Andrew Dingwall to Matthew Slattery for the modENCODE project.

value: Embryo 0-8

value: exd genomic coding region with stop codon replaced with GFP


title: cmi_0-8h_embryonic_ChIP-seq_ChIP_Rep1


title: cmi_0-8h_embryonic_ChIP-seq_Input_Rep1

description: input DNA


title: CG8478_S2_cells_ChIP-seq_ChIP_Rep1

description: ChIP DNA; Antibody information listed below: official name: KW3-CG8478-D1;target name: CG8478;host: Rabbit;antigen: MKAQLPKIGGTSGINHPMTMPQKPQQRISTIPKLAPSQGTQSMAPPSVKKIPKNVAHAGVYRTPTKTVPCHICKQSFRSILEFTNHSLTVHGNNQLKKMTGREDAQSAHDC;clonal: Polyclonal;purified: Protein A/G;company: Other;catalog: Aviva Systems Biology;short description: This is a custom made polyclonal antibody against CG8478 generated by the White Lab for the modENCODE project.

value: embryo-derived cell-line

value: late embryonic stage


title: CG8478_S2_cells_ChIP-seq_Input_Rep2

description: input DNA


title: CG8478_S2_cells_ChIP-seq_ChIP_Rep2


title: H3K4me1_W3L_ChIP-seq_ChIP_Rep1

value: Unknown


title: seq-SDQ5413_CEC7_fem2_AD_ChIP_Rep2

description: ChIP DNA; Antibody information listed below: official name: Y37D8A.11 SDQ5413;target name: CEC-7;host: Rabbit;antigen: ÔªøEAPKETVQKKARIEPVDSAEESKAKGSTAPMRKSAKKVGESNSEATDPEAKAKESSQSALKVEPPLTTHNSSRKTVEATGAMKRLTVAKETEDEALKSQKKARIEPVEDASKAKGSAAPKSASSRAEDGESSSTNDAAGTR;clonal: Polyclonal;purified: Affinity;company: SDI;catalog: SDQ5413;short description: An Affinity purified Rabbit Polyclonal antibody to CEC-7 obtained from SDI (Y37D8A.11 SDQ5413)


title: Abd-B-GFP_W3L_ChIP-seq_ChIP_Rep2

value: Abd-B-GFP

value: Abd-B genomic coding region with stop codon replaced with GFP


title: Abd-B-GFP_vMock_W3L_ChIP-seq_ChIP_Rep2


title: Abd-B-GFP_W3L_ChIP-seq_Input_Rep1

description: input DNA


title: Abd-B-GFP_W3L_ChIP-seq_Input_Rep2

description: input DNA


title: Abd-B-GFP_W3L_ChIP-seq_ChIP_Rep1


title: Abd-B-GFP_vMock_W3L_ChIP-seq_ChIP_Rep1


title: Abd-B-GFP_vMock_W3L_ChIP-seq_Input_Rep2

description: input DNA


title: H3K4me3_WPP-seq_ChIP_Rep2

extract_protocol:
Collection of White pre-pupae for chromatin extraction
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911).  After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.

value: White Prepupae (WPP)


title: H3K4me1_S2_cells_ChIP-seq_Input_Rep2

description: input DNA


title: H3K4me1_S2_cells_ChIP-seq_Input_Rep1

description: input DNA


title: Abd-B-GFP_vMock_W3L_ChIP-seq_Input_Rep1

description: input DNA


title: seq-ab817_8WG16_639746_N2_Eemb_Input_Rep2

description: input DNA

extract_protocol:
Worm_embryo_extraction_vSS2. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl) + protease inhibitors. Sample was dounced 30 times using tight pestle at 4ºC, then sonicated 9 times on ice at the following settings: 30 sec; power output 3; 70% duty cycle. Cell debris was removed by centrifuging at 13,000 rpm for 15 minutes at 4ºC and taking the supernatant. Supernatants were filtered through Millipore Ultrafree-MC 0.45 ?m filter units (cat. UFC30HV0S) at 13,000 rpm, 4°C for 1 minute to remove lipids. Protein concentration was determined, and extracts were aliquoted and stored at -80ºC. For a detailed protocol see http://www.modencode.org/.
Worm_chromatin_immunoprecipitation_vSS4. 0.3-3 mg extract + 1% sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl). Antibody was added to each IP sample and incubated overnight at 4ºC. Immune complexes were incubated (2 hrs at 4ºC) with 50 ?l of IgG dynabeads (Dyna), and washed 5 minutes with 1.5 mL of each of the following solutions: ChIP Buffer, ChIP Buffer + 1 M NaCl, ChIP Buffer + 500 mM NaCl, TEL Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and 2X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Samples were eluted twice with 150 ?L elution buffer for 15 minutes at 65ºC. Samples were treated with RNAse for 30 minutes at 37ºC. Samples were treated with proteinase K at 55ºC for 1-2 hrs then transfer to 65ºC overnight to reverse crosslinks. DNA was cleaned up using Zymo kit. For a detailed protocol see http://www.modencode.org/.
Worm_ChIP-seq_ DNA_Library_Prep_vSS2. ChIP and 10ng of input are blunted (T4 polymerase), an A-overhang is added (Klenow), DNA is ligated (concentrated T4 ligase) to annealed adaptors, amplified by PCR, and size-selected to the 300-400 bp range. Changes: Add AMPure cleanup after ligate adapter step. During size selection, increase run time to 3 hrs and decrease voltage to 80V. Increase size selection to at least 300 bp.


title: H3K4me3_WPP-seq_Input_Rep1

description: input DNA


title: H3K4me3_WPP-seq_ChIP_Rep1


title: H3K4me3_WPP-seq_Input_Rep2

description: input DNA


title: cmi_0-8h_embryonic_ChIP-seq_Input_Rep2

description: input DNA


title: USP-GFP_0-12-seq_Input_Rep1

description: input DNA

extract_protocol:
1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder.2. For a 0-12 hours egg laying, new plates are added after a 2hours pre-egglaying at 9pm and flies are allowed to lay eggs for 12 hours.3. Embryos are collected at 8.45 am using a filter mesh and a brush. They are rinsed with EWB and cross-linked at 9am in 1.8% formaldehyde at room temperature for 15 min.
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911).  After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.

value: USP-GFP

value: Embryo 0-12h

value: USP genomic coding region


title: USP-GFP_0-12-seq_Input_Rep2

description: input DNA


title: USP-GFP_WPP+12-seq_ChIP_Rep3

extract_protocol:
1. y; bw cn sp flies are cultivated in standard fly food covered with yeast powder.2. Flies are removed from the bottles after 24 hours and eggs are allowed to grow at 25 C for several days until they reach the desired growth stage.3. Pupae are collected from the side of the bottles using a brush 12 hours after the puparium formatin. 4. They are rinsed with EWB and cross-linked in 1.8% formaldehyde at room temperature for 15 min.chromatin collection is performed as described in The ChIP-chip protocol from the White lab.
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911).  After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.


title: USP-GFP_0-12-seq_ChIP_Rep1


title: CG8478_S2_cells_ChIP-seq_Input_Rep1

description: input DNA

value: Male


title: USP-GFP_WPP+12-seq_ChIP_Rep2


title: seq-JL00006_ZFP1_N2_L3_ChIP_Rep1

description: ChIP DNA; Antibody information listed below: official name: JL00006 ZFP1;target name: ZFP-1;host: Rabbit;antigen: ISQNGTSSTSQSNRLNLPSFMEQLLERQ WDQGSSLLMANAHFDVAQLLSCLFQLKSENFRLEENLSGLRKRRDHLFALNSRLAEVNTLDVSKRQRSDGLLLQQQIAHHLDPTSIVPKAEVHKQEPLSA PTSVPLPANHSSSLFEDIKAPKATYSRKTPSNIPLTVPLSTAATALTTTTAASSGAPVNSNIQNHRATPSTAGAPMAATPIMTAVTSANELAALSPERAQ ALLNMYRMPLDANVAAQLSMITNFPGQVNPSLFSRLLAVNMMNGGLQPNGQPLSALPPPTSATPNGK;clonal: Polyclonal;purified: Affinity;company: Covance;catalog: Jason Lieb lab;short description: A rabbit polyclonal antibody raised against amino acids 573-867 of AAK26137/WP:CE25003/F54F2.2a. Antibody was affinity purified against the same region of ZFP-1 as used for immunization.

data_processing:
Worm popcorns were grinded using a mixer mill 400 MM (Retsch) and fixed by 1% formaldehyde for 10 min at room temperature. After quenching the reaction, samples were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate; 150 mM NaCl) supplemented with protease inhibitors, phosphatase inhibitors and 1% sarkosyl. Using a Bioruptor water-bath sonicator (Diagenode), samples were sonicated at 4 oC with the following settings: ?High? amplitude, 30 sec on/30 sec off, 15 min (including offs). After separating the supernatant from the pellet by centrifugation, the pellet was again sonicated at the same condition but in a half volume of the buffer. Supernatants from the two sonication steps were combined, aliquoted and stored at -80C. For a detailed protocol see http://www.modencode.org/.
DNA was blunt-ended with End Repair Enzyme mix (Epicentre, Madison) and A-overhanged with Exo(-) Klenow fragment in the presence of dATP. DNA fragments were ligated with adaptors. DNA was amplified by PCR with single-end or pair-end primers. Amplicon was loaded on an agarose gel, and DNA at an indicated size range was recovered from the gel.
ChIP-seq_alignment-BWA:JL:1 protocol. BWA is a fast light-weighted tool that aligns relatively short sequences to a sequence database. The algorithms is based on Burrows-Wheeler Transform (BWT). This protocal, was ran as DEFAULT for both bwa aln and bwa samse.bwa aln: Find the SA coordinates of the input reads. Maximum maxSeedDiff differences are allowed in the first seedLen subsequence and maximum maxDiff differences are allowed in the whole sequence.OPTIONS:-n NUM
Maximum edit distance if the value is INT, or the fraction of missing alignments given 2% uniform base error rate if FLOAT. In the latter case, the maximum edit distance is automatically chosen for different read lengths. [0.04]-o INT
Maximum number of gap opens [1]-e INT
Maximum number of gap extensions, -1 for k-difference mode (disallowing long gaps) [-1]-d INT
Disallow a long deletion within INT bp towards the 3?-end [16]-i INT
Disallow an indel within INT bp towards the ends [5]-l INT
Take the first INT subsequence as seed. If INT is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for ?-k 2?. [inf]-k INT
Maximum edit distance in the seed [2]-t INT
Number of threads (multi-threading mode) [1]-M INT
Mismatch penalty. BWA will not search for suboptimal hits with a score lower than (bestScore-misMsc). [3]-O INT
Gap open penalty [11]-E INT
Gap extension penalty [4]-R INT
Proceed with suboptimal alignments if there are no more than INT equally best hits. This option only affects paired-end mapping. Increasing this threshold helps to improve the pairing accuracy at the cost of speed, especially for short reads (~32bp).-c
Reverse query but not complement it, which is required for alignment in the color space.-N
Disable iterative search. All hits with no more than maxDiff differences will be found. This mode is much slower than the default.-q INT
Parameter for read trimming. BWA trims a read down to argmax_x{\sum_{i=x+1}^l(INT-q_i)} if q_l<INT where l is the original read length. [0] bwa samse:Generate alignments in the SAM format given single-end reads. Repetitive hits will be randomly chosen.OPTIONS:-n INT
Maximum number of alignments to output in the XA tag for reads paired properly. If a read has more than INT hits, the XA tag will not be written. [3]  Processed data are obtained using following parameters: genome version is ce10   ChIP-Seq_MACS_Peak-calling:JL:1 protocol. Peak calls were determined by using Model-based Analysis of ChIP-Seq (MACS) software. MACS was supplied with the following parameters:tagSize = 25mfold = 10,30genomeSize = 90000000bandwidth = 300pvalue = 1e-5  Processed data are obtained using following parameters: genome version is ce10

extract_protocol:
Worm_L3_extraction_vKI1.doc
Worm_chromatin_immunoprecipitation_v5. 2mg extract + 1%sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl) and treated with RNAse. Antibody was added to each IP sample and incubated overnight at 4C. Immune complexes were incubated (2hrs at 4C) with 50 ul of IgG dynabeads (Dyna), and washed 5 minutes with 1.5mL of each of the following solutions: ChIP Buffer, ChIP Buffer+1M NaCl, ChIP Buffer+500mM NaCl, TEL Buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA), and 2X TE (10mM Tris-HCl pH 8.0, 1mM EDTA). Samples were eluted twice with 150uL elution buffer for 15minutes at 65C. Samples were treated with proteinase K at 55C for 1-2hrs then transfer to 65C overnight to reverse crosslinks. DNA was cleaned up Qiagen PCR purification kit and concentrated using by Speed vac.
Solexa_Library_Prep_vKI3
Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.

value: L3 Larva

value: wild type


title: USP-GFP_WPP+12-seq_Input_Rep3

description: input DNA


title: USP-GFP_0-12-seq_ChIP_Rep2


title: USP-GFP_WPP+12-seq_Input_Rep2

description: input DNA


title: USP-GFP_WPP+12-seq_Input_Rep1

description: input DNA