title: GSM8457506: ChIRPseq_wt_2; Arabidopsis thaliana; OTHER
library_construction_protocol: 3'RNA-seq: After grinding in liquid nitrogen, samples were resuspended in 500 μl of homogenisation buffer (100 mM Tris pH 8.5, 5 mM EDTA, 100 mM NaCl, 0.5% SDS, 1% β-mercaptoethanol (Sigma)) and centrifuged for 5 min at RT at 8000 g. The supernatant was transferred to the new tubes containing 250 μl of chloroform. After 15 min of shaking, 250 μl of phenol was added, and shaking was repeated, followed by centrifugation for 10 min at 10000 g. The upper phase was transferred to new tubes, and an equal volume of phenol:chloroform:isoamyl alcohol was added. Shaking and centrifugation steps were repeated. The upper phase was mixed with 10% of the volume of 3 M sodium acetate (pH 5.2), and an equal volume of isopropanol was added. After centrifugation for 30 min at 20000g at 4°C the pellet was washed with 70% EtOH and resuspended in water. ChIRP-seq and ChIDP-seq: To uncover RNA-DNA (Chromatin Isolation by RNA Purification) and DNA-DNA (Chromatin Isolation by DNA Purification Assay) interactions, we used 400 mg of fresh siliques (13-20 days after pollination), which were ground in liquid nitrogen and resuspended in 20 ml of crosslink buffer (10 mM HEPES-KOH pH 7.4, 50 mM NaCl, 100 mM sucrose, 1% formaldehyde (for RNA-RNA and RNA-DNA) or 0.5% glutaraldehyde (for ChIDP)). After 10 min incubation on a rotator at 4°C, the crosslinking reaction was quenched by adding 1.25 ml 2 M glycine. After an additional 5 min incubation on a rotator, samples were centrifuged at 4°C and 2,000 g for 15 min. The pellets were resuspended in 20 ml of cold Honda Buffer (20 mM HEPES-KOH pH 7.4, 0.44M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 10 mM MgCl2, 5 mM DTT, 0.5% Triton X-100, 10 mM β mercaptoethanol, 1 mM PMSF, 1x Complete protease inhibitors (Roche) and 5 U/ml murine RNase inhibitor (NEB)) and filtered through Miracloth. Subsequently, samples were centrifuged for 15 min at 2,000 g at 4°C. According to downstream assays, the nuclei pellets were resuspended in appropriate buffers (Element Zero Biolabs kit). To enrich specific RNA molecules or chromatin fragments, we used MagIC beads with custom probes targeting those sequences (Element Zero Biolabs). Element Zero Biololabs designed probes are delivered with kits and remain confidential as protected intellectual property. For ChIRP-seq with MUSHER and lacZ probes, nuclei pellets were resuspended in 1.1 ml of MagIC Lysis Buffer FA (Element Zero Biolabs kit), supplemented with 1x Complete protease inhibitors (Roche) and 5 mM DTT. Next, samples were sonicated using a Bioruptor (Diagenode) and diluted two times with MagIC Lysate Dilution Buffer FA, with 1% of the volume saved as an input sample. MagIC beads were pre-cleared according to the producers' protocol and added to the samples that were next incubated at 40°C for 30 minutes with occasional shaking. This was followed by three washes using MagIC Wash Buffer I FA and nucleic acids elution by incubation at 92°C for 5 min with 10 mM Tris-HCl pH 7.5. After Proteinase K treatment (55°C, 90 min) and phenol:chloroform:isoamyl alcohol extraction, DNA was precipitated by adding 1 µl glycogen, 10% of the volume of 3 M LiCl, and an equal volume of isopropanol. Samples were centrifuged for 30 min at 4°C at 20,000 g. Pellets were washed with cold 80% ethanol and resuspended in water. For ChIDP-seq, the nuclei pellet was resuspended in 1.1 ml of MagIC Lysis Buffer FA (Element Zero kit) supplemented with 1x Complete protease inhibitors (Roche) and 5 mM DTT. Samples were sonicated using Bioruptor (Diagenode), diluted with MagIC Lysate Dilution Buffer FA, and split into halves (treated with RNaseA at 37°C for 20 min and non-treated). MagIC beads recognising the PIR1 locus were mixed with the lysates that were next incubated at 81°C for 5 min with occasional shaking to denature DNA partially. Subsequently, samples were transferred to 40°C for 20 min to enable binding of the probes. This was followed by six washing steps with the MagIC Lysate Wash buffer I and II, and DNA was eluted with 10 mM Tris-HCl pH 7.5 at 92°C for 5 min. After adding an equal volume of MagIC Lysate Wash buffer I to eluted samples, proteinase K treatment (55°C, 120 min) was performed. To extract DNA fragments, an equal volume of phenol:chloroform:isoamyl alcohol was added. The upper phase was mixed with 1 µl of glycogen, 10% of the volume of 3 M LiCl and an equal amount of isopropanol. After centrifugation for 30 min at 20000g at 4°C, the pellet was washed with 80% EtOH and resuspended in water. 3'RNA-seq: 500 ng of RNA was used in reverse transcription with 50 mM different barcoded oligo(dT) primers and SuperScript III. 0.25 of each reaction was pooled and pools were purified with Ampure beads (1.2x beads to sample volumes) and eluted in 17 ul of water. 15 ul of recovered cDNA pool was mixed by pipetting with 15 ul of second-strand synthesis mix (2x NEBNext Second Strand Synthesis (dNTP-free) Reaction Buffer (NEB), 1 U RNaseH (NEB), 1 U E. coli DNA ligase I (NEB), 5 U E. coli DNA polymerase (NEB), 30 uM dNTPs) and incubated overnight at 16°C. Reactions were purified with Ampure beads (1.2x beads to sample volumes) and eluted in 4 ul of water. 3 ul of recovered dsDNA sample was used for tagmentation using 3 ul 5x diluted B-loaded Tn5 enzyme (homemade) and 6 ul freshly prepared 2x buffer (20 mM Tris-HCl pH 7.5, 20 mM MgCl2, 50% DMF). Reactions were mixed by pipetting and incubated for 7 min at 55°C followed by 5 min at 80°C. Water was added up to 30 ul, samples were purified with Ampure beads (1.2x beads to sample volumes) and eluted with 11 ul of water. Illumina indexing PCR was performed using 10 ul tagmented DNA in 50 ul reactions with Q5 2× PCR Master Mix (NEB) and 0.5 uM final concentration of primers. Libraries were Illumina Next-seq sequenced to obtain two reads: 21 nt R1 (contain barcode and UMI) and 55 nt R2 (contain mRNA sequence). For ChIRP-seq and ChIDP-seq, DNA fragments eluted from MagIC beads were used to prepare libraries with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina using NEBNext Multiplex Dual Index Primers.