title: GSM4729223: HUVEC_H_T7d_2_iMARGI; Homo sapiens; OTHER
library_construction_protocol: Approximately 5 million cells were used for construction of an iMARGI sequencing library. Cells were crosslinked in 1% formaldehyde at room temperature (RT) for 10 minutes with rotation. Crosslinking reaction was quenched with glycine at 0.2 M concentration and incubated at RT for 10 minutes. Cells were pelleted, washed using 1 × PBS and aliquoted into ~ 5 million in each tube. To prepare nuclei, crosslinked cells was incubated in 1 mL of cell lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% Nonidet P-40, 1× protease inhibitor) on ice for 15 minutes and homogenized with dounce homogenizer pestle A for 20 strokes on ice. Nuclei were pelleted and weighed to estimate the pellet volume (10 mg of nuclei pellet was estimated to be about 10 µL). Nuclei pellet were incubated with SDS buffer (0.5× Cutsmart buffer, 0.5% SDS) with 1:3 volume ratio at 62 °C for 10 minutes with mixing and immediately quenched with a final of 1% Triton X-100. To digest chromatin, washed nuclei pellet was re-suspended in AluI chromatin digestion mix (2.3 U/µL AluI (NEB), 0.3 U/µL RNasinPlus, 1× Cutsmart buffer) and incubated at 37 °C overnight with mixing. After chromatin digestion, 1 µL of RNase I (1:10 diluted in 1× PBS) was directly added to the reaction mixture and incubated at 37 °C for 3 minutes to fragment RNA. Nuclei were pelleted and washed twice using PNK wash buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2). To prepare the RNA and DNA ends for linker ligation, nuclei were incubated in 200 µL RNA 3´ end dephosphorylation reaction mix (0.5 U/µL T4 PNK (NEB), 0.4 U/µL RNasinPlus, 1× PNK phosphatase buffer pH 6.5) at 37°C for 30 minutes with mixing. Nuclei were washed twice with PNK buffer, resuspended in 200 µL DNA A-tailing mix (0.3 U/µL Klenow fragment 3´-5´ exo- (NEB), 0.1 mM dATP, 0.1% Triton X-100, 1× NEB buffer 2) and incubated at 37°C for 30 minutes with mixing. The same linker sequence as described in the previous MARGI paper was used. For in situ RNA-linker ligation, nuclei were resuspended in 200 µL ligation mix (38 µL adenylated and annealed linker, 10 U/ µL T4 RNA ligase 2-truncated KQ (NEB), 1× T4 RNA ligase reaction buffer, 20% PEG 8000, 0.1% Triton X-100, 0.4 U/µL RNasinPlus) and incubated at 22°C for 6 hour and then 16°C overnight with mixing. After ligation, the nuclei were washed five times with PNK buffer to remove excess free linker. For in situ RNA-DNA proximity ligation, nuclei were resuspended in 2 mL of proximity ligation mixture (4 U/µL T4 DNA ligase (NEB), 1× DNA ligase reaction buffer, 0.1% Triton X-100, 1 mg/mL BSA (NEB), 0.5 U/µL RNasinPlus) and incubated at 16 °C overnight. To reverse crosslinking, nuclei were washed twice with 1× PBS, resuspended in 250 µL of extraction buffer (1 mg/mL Proteinase K [NEB], 50 mM Tris-HCl pH 7.5, 1% SDS, 1 mM EDTA, 100 mM NaCl) and incubated at 65 °C for 3 hours. DNA and RNA were extracted by adding an equal volume of Phenol:Chloroform:Isoamyl Alcohol (pH 7.9, Ambion) followed by ethanol precipitation. The subsequent steps including removal of biotin from non-proximity ligated linkers, pulldown of RNA-DNA chimera, reverse transcription of RNA, DNA denaturation, circularization, oligo annealing and BamHI (NEB) digestion and sequencing library generation were performed as previously described. iMARGI libraries were subsequently subject to pair-end 100-cycle sequencing on Illumina HiSeq 4000. The circularization and library construction strategy can phase RNA and DNA ends into Read 1 and Read 2. Since AluI restriction enzyme recognizes "AGCT" sequence and leaves "CT" at the 5´ end of the cut, we expect the first two bases of DNA end (Read 2) to be enriched with "CT".