title: GSM1145577: zfp-1 mutant, 2nd replicate; Caenorhabditis elegans; OTHER
library_construction_protocol: Worms were washed off the plate with cold M9 and washed 3-4 times.  The worm pellet was resuspended in 2ml of cold Nuclear Run-On (NRO) lysis buffer (10mM Tris pH 7.5, 2mM MgCl2, 3mM CaCl2, 0.5% IGEPAL and 10% glycerol), transferred to a steel dounce (on ice) and stroked 20 times.  The worm lysate was centrifuged for 2min at 40g and the supernatant was centrifuged again for 5min at 1000g to pellet the nuclei.  Next, the supernatant was discarded and the nuclei pellet was washed two times with 1ml of cold NRO lysis buffer and one time with 1ml of cold Freezing Buffer (50mM Tris-CL pH 8.3, 40% glycerol, 5mM MgCl2, 0.1 mM EDTA). Nuclei were then resuspended in 100µl of Freezing Buffer and used for the NRO reaction. //  NRO reaction and DNase digestion: 100µl of nuclei were mixed with an equal volume of reaction buffer (10mM Tris-Cl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCL, 20 units of SUPERase In, 1% sarkosyl, 500µM ATP, GTP, and Br-UTP, 2μM CTP) and incubated for 5 minutes at 32°C.  The reaction was stopped with 750μl of TRIzol reagent (Invitrogen) and the RNA was extracted following the manufacturer's instructions.  The RNA pellet was resuspended in 85µl water and treated with 5µl of turbo DNase (Ambion) in a total volume of 100µl and incubated at 37°C for 30 minutes.  The RNA was then extracted by phenol:chloroform and precipitated with one volume of isopropanol with 1/10 volume of Sodium Acetate 3M and 10µg of Glycoblue (Ambion).  // Base Hydrolysis of RNA: The hydrolysis of RNA was performed using fragmentation reagent (Ambion) for 15min at 70°C and the product was purified by p-30 RNase free spin column (BioRad), following the manufacturer’s instruction. //  Immunopurification of Br-U RNA and RNA-end repair: The purification of the Br-U RNA and the end repair of the purified Br-U RNA was performed as described in (Core et al., 2008). Adapter ligation and library preparation:  For the ligation of 5' and 3' adapters to Br-U RNA we used reagents contained in the ScriptMiner™ Small RNA-sequencing kit (Epicentre) and we immunopurified the ligated Br-U RNA product after each ligation with anti-deoxy-BrU beads (Santa Cruz Biotech (sc-32323-AC).  The ligated Br-U RNA was then reverse-transcribed and PCR-amplified with properly-indexed primers for 18 cycles, following the ScriptMiner™ Small RNA-sequencing kit (Epicentre) manufacturer's instructions.  The NRO-cDNA libraries were purified by phenol:chloroform, the DNA was precipitated as above and then ran on a non-denaturing 1XTBE, 8% acrylamide gel and stained with SYBR gold (Invitrogen). DNA fragments ranging in size from 120bp to 300bp was excised from the gel and eluted by incubating in TE + 300mM NaCl overnight with rotation.  The DNA libraries were then extracted, precipitated, and sent for sequencing.