title: GSM8618297: WT-DMSO-pNET-rep1; Arabidopsis thaliana; ncRNA-Seq
library_construction_protocol: Seedlings were harvested from 1/2MS liquid media and were grounded in liquid nitrogen and resuspended in ice-cold lysis buffer (50mM HEPES pH 7.5, 150mM NaCl, 1mM EDTA pH 8.0, 1% Triton X-100, 10% glycerol, 5mM β-mercaptoethanol, 1mM PMSF, 2μg/μl Aprotinin and 2μg/μl Pepstatin A) for 30 mins on ice. After lysis mixture was filtered and centrifuged to discard the supernatant, Nuclear pellet was washed once with ice-cold HBB (25mM Tris-HCl pH7.6, 0.44M sucrose, 10mM MgCl2, 0.10% Triton-100, 10mM β-mercaptoethanol) and HBC (20mM Tris-HCl pH7.6, 0.352M sucrose, 8mM MgCl2, 0.08% Triton-100, 8mM β-mercaptoethanol and 20% glycerol) buffers, respectively. Washed nuclear pellet was resuspended in MNase reaction buffer (20mM Tris-HCl pH 8.0 , 5mM NaCl, and 2.5mM CaCl2, 20U MNase (NEB)). The reaction mixture was incubated at 37°C for 5 minutes and the reaction was later stopped with final con. 0.02M EDTA. Then sonication was performed 1s ON/OFF for 10 cycles to release chromatin and centrifuged to obtain the supernatant. The collected supernatant was diluted 5-8 times with ice-cold NET-2 buffer (50mM Tris-HCl pH7.4, 150mM NaCl, and 0.05% NP-40). The diluted supernatant was incubated with Pol II Ser2p antibody (ab5095) coated Dynabeads (Invitrogen, 11201D) for 2 hours at 4°C and the beads containing complex was washed 8 times with ice-cold NET-2 buffer. Finally, beads were resuspended with PNK reaction mixture containing 75 μl PNK buffer, 10 μl PNK enzymes (NEB) and 15 μl of 10mM ATP and incubated at 37°C for 10 mins on a Thermomixture (1400 rpm). After that supernatant was removed and beads were washed once with NET-2 buffer. Then beads was followed RNA extraction with Trizol reagent. The extracted RNA was conducted to library construction. The recovered RNA was used to construct the small RNA libraries with NEXTflex TM small RNA-Seq Kit V4. Finally, the libraries were recovered from the 6% PAGE gel corresponding to 140-250 bp size and undergone high throughput deep sequencing. small RNA-seq