Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: ChIP-seq experiments (sample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21): 100-200 μl frozen synchronized young adult worm pellets were used for each chromatin immnuoprecipitation experiment. Crushed pellets (pulverized by grinding in liquid nitrogen with a mortar and pestle) were resuspended in 1ml of pre-chilled RIPA buffer (1X PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1X HALT combined protease and phosphatase inhibitor cocktail [ThermoScientific]). To crosslink, formaldehyde was added to the crude extract to a final concentration of 2%. The lysate was rotated at 4°C for 10 minutes. 0.1 ml of 1M Tris-HCl (pH 7.5) was added to quench formaldehyde. Lysate was spun at 6000 x g for 30 second and the supernatant was removed. The pellet was resuspended in 0.5ml of pre-chilled RIPA buffer. The lysate was transferred to a 1.5 ml TPX tube (Diagenode) and sonicated (Bioruptor [Diagenode], output level: high, interval: 0.5, three times of 8-minute sonication). 50 μl of the lysate was used to make IP input library. For each immumoprecipiation experiment, 2 μg of anti-H3K9me3 (ab8898, Abcam), anti-Pol II-8WG16 (ab817, Abcam), anti-Pol II-S2 (ab5095, Abcam), or anti-Pol II-S5 (ab5131, Abcam) antibody was added to 400 μl of the lysate (containing approximately 50 μg DNA). The IP mix was rotated overnight at 4°C. 50 μl of Protein A Dynabeads (Life Technology) was added and rotated for another 2 hours. The beads were then washed three times with 800 μl ice-cold LiCl washing buffer (100 mM Tris-HCl, pH7.5, 500 mM LiCl, 1% NP-40 and 1% sodim deoxycholate). To elute the immunoprecipitation product and reverse crosslink, beads were incubated with 400 μl of worm lysis buffer (0.1 M Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, and 200 μg /ml of proteinase K) at 65 °C for 2 hours with agitation every 30 minutes (IP input lysate were treated similarly to reverse crosslink) and then subject to organic extraction and DNA precipitation. small RNA-seq experiments (sample 18 and 19): Small RNA was extracted from frozen synchronized adult worm pellets (100-200 μl) using mirVana miRNA isolation kit (Life technologies). pre-mRNA-seq experiments (sample 11 and 12): Worm grinds were resuspended in 1ml of pre-chilled RIPA buffer (1xPBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1X HALT combined protease and phosphatase inhibitor cocktail [ThermoScientific]). To cross-link, formaldehyde was added to the crude extract to a final concentration of 2%. The lysate was rotated at 4 °C for 10 minutes. 0.1 ml of 1M Tris-HCl (pH 7.5) was added to quench formaldehyde. Lysate were spun at 6000 x g for 30 second and the supernatant was removed. The pellet was resuspended in 0.5ml of pre-chilled RIPA buffer. The lysate was transferred to a 1.5 ml TPX tube (Diagenode) and sonicated (Bioruptor [Diagenode], output level: high, interval: 0.5, three times of 8-minute sonication). For each immumoprecipiation experiment, 2 μg anti-Pol II-S2 (ab5095, Abcam) antibody was added to 400 μl of the lysate (approximately 50 μg DNA). The IP mix was rotated overnight at 4 °C . 50 μl of Protein A Dynabeads (Life Technology) was added and rotated for another 2 hours. The beads were then washed three times with 800μl ice-cold LiCl washing buffer (100 mM Tris-HCl, pH7.5, 500 mM LiCl, 1% NP-40 and 1% sodim deoxycholate). To elute the immunoprecipitation product and reverse corss-links, beads were incubated with 400 μl of worm lysis buffer (0.1 M Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, and 200 μg /ml of proteinase K) at 65 °C for 2 hours with agitation every 30 minutes (IP input lysate were treated similarly to reverse cross-link samples) and then subject to phenol/Chloroform extraction, RNA/DNA precipitation, DNase I treatment, phenol/Chloroform extraction, and RNA precipitation. RNA was then fragmentated using Fragmentation Reagents (Ambion). mRNA-seq experiments (sample 13, 14, 15, 16, and 17): Total RNA was extracted from frozen synchronized adult worm pellets (100-200 μl) using Trizol (Life Technologies). mRNA was selected from total RNA using Poly(A)Purist MAG kit (Life Technologies). mRNA was then fragmentated using Fragmentation Reagents (Ambion). 0.5-1 μg of mRNA was used for each mRNA-seq library. ChIP-seq (sample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21): Same as described in Gu SG et. al. 2012 (PMID: 22231482). pre-mRNA-seq (sample 11 and 12): The fragmented RNA was treated with T4 polynucleotide kinase (New England Biolabs). The 3’ end of RNA was ligated to an adenylated DNA oligo (IDT linker-1: 5’-rAppCTGTAGGCACCATCAATC-3’) using T4 RNA ligase 1 (New England Biolabs) without ATP. ~15-35 nt RNA captured by the 3’ linker oligo was purified using 8 % polyacrylamide (acrylamide:bis-acrylamide = 19:1) gels containing 8 M urea, followed by a 5’ end ligation reaction using a DNA-RNA hybrid oligo (5’-ACGCTCTTCCGATCTrNrNrNrN-3’, rNrNrNrN as 4-nt barcode) using T4 RNA ligase 1 (New England Biolabs) with ATP. cDNA was synthesized by using SuperScript III Reverse transcriptase (Life Technologies) and primer SG460 (5‘-GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3’), followed by PCR amplification with primers SG-465 (5‘-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and SG-46X (5’-CAAGCAGAAGACGGCATACGAGAT[6-nt index]GTGACTGGAGTTCAGACGTGTGCTCTTCC-3’). To ensure that amplified DNA are still annealed to a true complement and avoid reannealing-distortion in the resulting libraries ( referenceParameswaran et al., 2007), PCR reactions were titrated with increasing cycle numbers; cycle numbers for which product levels have not saturated (i.e. product levels still able to increase substantially with additional cycles) were selected. After separating PCR products on 3 % agarose gels, DNA bands of the expected size were extracted (QIAchange® Gel Extraction Kit [Invitrogen]; omitting the 50°C heating step), followed by massive parallel DNA sequencing (Illumina HiSeq2000). small RNA-seq (sample 18 and 19): Same as described in Gu SG et. al. 2012 (PMID: 22231482). mRNA-seq (sample 13, 14, 15, 16, 17): 0.5-1 μg fragmented oligo(dT)-selected mRNA was used to construct each mRNA-seq library. The fragmented RNA was treated with T4 polynucleotide kinase (New England Biolabs). The 3’ end of RNA was ligated to an adenylated DNA oligo (IDT linker-1: 5’-rAppCTGTAGGCACCATCAATC-3’) using T4 RNA ligase 1 (New England Biolabs) without ATP. ~35-60 nt RNA captured by the 3’ linker oligo was purified using 8 % polyacrylamide (acrylamide:bis-acrylamide = 19:1) gels containing 8 M urea, followed by a 5’ end ligation reaction using a DNA-RNA hybrid oligo (5’-ACGCTCTTCCGATCTrNrNrNrN-3’, rNrNrNrN as 4-nt barcode) using T4 RNA ligase 1 (New England Biolabs) with ATP. cDNA was synthesized by using SuperScript III Reverse transcriptase (Life Technologies) and primer SG460 (5‘-GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3’), followed by PCR amplification with primers SG-465 (5‘-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and SG-46X (5’-CAAGCAGAAGACGGCATACGAGAT[6-nt index]GTGACTGGAGTTCAGACGTGTGCTCTTCC-3’). To ensure that amplified DNA are still annealed to a true complement and avoid reannealing-distortion in the resulting libraries ( referenceParameswaran et al., 2007), PCR reactions were titrated with increasing cycle numbers; cycle numbers for which product levels have not saturated (i.e. product levels still able to increase substantially with additional cycles) were selected. After separating PCR products on 3 % agarose gels, DNA bands of the expected size were extracted (QIAchange® Gel Extraction Kit [Invitrogen]; omitting the 50°C heating step), followed by massive parallel DNA sequencing (Illumina HiSeq2000). ChIP-seq for sample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21. small RNA-seq (5' monophosphate-independent) for sample 11,12,18 and 19. mRNA-seq for sample 13, 14, 15, 16,