GSM2304927: pre-mRNA-seq_hrde-1_OP50; Caenorhabditis elegans; RIP-Seq
100-200 μl frozen synchronized young adult worm pellets were used for each pre-mRNA-seq 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). For each immumoprecipiation experiment, 2 μg of anti-Pol II-S2 (ab5095, Abcam) 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 1.5-2 hours with agitation every 30 minutes and then subject to organic extraction and precipitation of total DNA and RNA. DNA/RNA mix was treated with DNase I (NEB) and resulted RNA was used for the pre-mRNA-seq library preparation. pre-mRNA library was prepaed using the following protocol: RNA was fragmented by hydrolysis using Fragmentation Reagent Kit (Ambion). RNA was treated with T4 PNK (NEB). Then RNA 3’ end was ligated to IDT linker-1 (5’-rAppCTGTAGGCACCATCAATC-3’) using T4 RNA ligase 2, truncated (NEB, M0242S). RNA library of about 35-60 nt was size selected using 8 % polyacrylamide (acrylamide:bis-acrylamide = 19:1) gels containing 8 M urea (size selection also eliminated excess IDT linker-1). RNA 5’end was ligated to DNA-RNA hybrid oligo (5’-ACGCTCTTCCGATCTrNrNrNrN-3’, where rNrNrNrN is a 4-nt barcode) using T4 RNA ligase 1 (NEB) with ATP. First strand cDNA was synthesized using SuperScript III RT kit (Life Technologies) and primer SG-460 (5’-GGAGTTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3’). cDNA library was PCR amplified at early cycle (tested by different cycle number to capture linear amplification phase and avoid over amplification). PCR was done using a common primer SG-465 (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and primer with 6-nt index for multiplexing (5’-CAAGCAGAAGACGGCATACGAGAT[ index]GTGACTGGAGTTCAGACGTGTGCTCTTCC-3’). Amplified library was size selected by 2% agarose gel and gel purified using QIAquick Gel Extraction Kit (QIAGEN).