characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2018-01-15T17:04:14Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2017-09-06T13:05:05Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA104285080
characteristics.characteristic: External Id
characteristics.text: The University of Texas at Dallas
characteristics.characteristic: INSDC center name
characteristics.text: 2018-01-15T17:04:14Z
characteristics.characteristic: INSDC first public
characteristics.text: 2017-09-06T13:05:05Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS1910058
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-6047:GM0h
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: GM12878
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0002784
characteristics.characteristic: cell line
characteristics.text: B cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000236
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: GM12878 cell line was obtained from Coriell Biorepository and maintained as recommended by the supplier. Cells were infected with Sendai Virus Cantell Strain (ATCC VR-907) from Charles River Laboratories was used. We infected cells at 3000 HA units per 1 million cells (or at ~1x10e8 EID50). To obtain nascent RNAs for sequencing, we followed the GRO-seq protocol from Greer et al, Cell Reports 13:1444-1455 (2015). Two 15-cm plates of confluent cells (~10-20 million cells) were washed 3 times with ice cold PBS buffer and incubated for 5 min with 10 mL cold swelling buffer (10 mM Tris-Cl pH 7.5, 2 mM MgCl2, 3 mM CaCl2) for each plate on ice. Cells were scraped from the plate, harvested, centrifuged at 500xg for 10 min at 4°C and were resuspended in 1 mL of lysis buffer (swelling buffer with 0.5% IGEPAL, 10% glycerol and 4 U/mL SUPERaseIn) with gentle mixing by pipetting with a widened boar pipette tip up and down 20 times. For the isolation of nuclei, 9 mL of the same lysis buffer (up to total 10 mL) was added. After collecting the nuclei by centrifugation (at 300xg for 5 min at 4°C), it was resuspended in 1 mL freezing buffer per 5 million nuclei, and pelleted and resuspended to the final volume of 100 μL (about 5-10 million nuclei/100 μL) of freezing buffer (50 mM Tris pH 8.3, 5 mM MgCl2, 0.1 mM EDTA, 40% glycerol). Before the NRO reaction, NRO reaction buffer (10 mM Tris-Cl pH 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 50 μM ATP, GTP and Br-UTP, 2 μM CTP, 0.4 U/μL RNasin, and 2% sarkosyl) was generated and pre-heated to 30°C for 5 min. An equal volume (100 μL) of NRO reaction buffer was mixed with 100 μL of thawed nuclei solution in freezing buffer and was incubated at 30 °C for 5 min with mixing at 800 rpm on a thermomixer. Then, RQ1 DNaseI (Promega) was added along with DNaseI reaction buffer and samples were incubated at 37 °C for 20 min with mixing 800 rpm. To stop the NRO reaction, 225 μL NRO stop solution was added to the reaction and 25 μL of Proteinase K was added. The sample was incubated for 1 hr at 55 °C. Nuclear RNA was extracted with acidic phenol (Sigma) then chloroform (Sigma), and was precipitated and washed. RNA was then resuspended in 20 μL of nuclease free water and subjected to base hydrolysis by addition of 5 μL of 1N NaOH on ice for 10 min. The reaction was neutralized with 50 μL of 0.5 M Tris-Cl pH 6.8. Then, RNA was purified through BioRad P-30 RNase-free spin column (BioRad) following to the manufacturer’s instructions, and was treated with 7 μL of DNaseI buffer and 3μL RQ1 DNaseI (Promega) for 10 min at 37 °C, and purified again with a BioRad P-30 column. Extracted RNA was then Br-UTP binding: BrU incorporated nascent RNA from total nuclear RNA was purified using Br-UTP beads.Anti-BrU agarose beads (Santa Cruz Biotech) were equilibrated by washing them 2 times in 500 μL BrU binding Buffer (0.25× SSPE, 1 mM EDTA, 0.05% Tween-20, 37.5 mM NaCl) and blocked in 1mL BrU blocking buffer (1x binding buffer, 0.1% PVP, and 1 mg/mL BSA) for 1 hr with rotation at 4 °C. During the blocking step, beads were washed 2 times with 500 μL binding buffer, NRO RNA sample was heated at 65 °C for 5 min then placed on ice at least for 2 min. 50 μL of the blocked bead mixture was combined with RNA sample in 450 μL binding buffer and combined with bind RNA to beads for 1 hr by rotating at 4 °C. After binding, beads were washed once in low salt buffer (0.2 × SSPE, 1 mM EDTA, 0.05% Tween-20), once in high salt buffer (0.25x SSPE, 1 mM EDTA, 0.05% Tween-20, 137.5 mM NaCl) and twice in TET buffer (TE with 0.05% Tween-20). BrU-incorporated RNA was eluted 4 times with 100 μL elution buffer (20 mM DTT, 300 mM NaCl, 5 mM Tris-Cl pH 7.5, 1 mM EDTA and 0.1% SDS). RNA was then extracted and precipitated as described above. The precipitated RNA was re-suspended in 20 μL of water. RNA was heated to 65°C for 5 min and cooled on ice at least for 2 min. The RNA was treated with TAP (by adding 3 μL 10x TAP buffer, 5 μL water, 1 μL SUPERaseIn (Promega), 0.5 μL TAP) at 37 for 1.5 hr, and then preincubated with PNK reaction premix (1 μL PNK(NEB), 1 μL 300 mM MgCl2, 1 μL 100 mM ATP) for 30 min. Afterward, PNK reaction main mix (20 μL PNK buffer (NEB), 2 μL 100 mM ATP (Roche), and 142 μL water, 1 μL SUPERaseIN (Promega) and another 2 μL PNK(NEB)) was added to the preincubated RNA sample and incubated at 37 °C for 30 min. The RNA was extracted and precipitated again as above, and resuspended in 9 μL water. 5’-adaptor ligation: BrU-RNA, 5’ adaptor (5uM) and PEG was heated on 65 °C for 5min then cooled on ice. Ligation mixture (1.5 μL 5’adaptor (5uM), 2uL 10x RNA ligation buffer, 1.5uL T4 RNA ligase, 1uL SUPERRNaseIN, 5uL 50% PEG 8000) was added to the 9uL BrU-RNA and incubated at 22 °C or RT for 4-6 hours. Then, 5’ adaptor ligated BrU-RNA was purified with the bead binding method as described above. 3’-adaptor ligation: the same ligation reaction for the 5’-adaptor ligation described above was performed with the 3’ adaptor in place of the 5’ adaptor. RT-reaction RNA and RT oligo (5’-CAAGCAGAAGACGGCATACGA-3’) was heated to 65 °C for 10 min and cooled on ice. RT reagent mixture (1 μL RT oligo (100 μM), 5x first strand buffer (Invitrogen), 10mM dNTPs (Roche), 100mM DTT (Invitrogen), 1 μL RNase inhibitor (Promega) without Superscript III (Invitrogen) was added to RNA sample and incubated at 48°C for 3 min, and then 1 μL superscript III was added to the RT reaction sample and incubated at 48°C for 20 min, 50°C for 45 min, sequentially. After RT reaction, RNA was eliminated by adding RNase cocktail and RNaseH and incubation at 37°C for 30 min. PCR amplification: The ssDNA template was amplified by PCR using the Phusion High-Fidelity enzyme (NEB) according to the manufacturer’s instructions. The small RNA PCR primers (5′-CAAGCAGAAGACGGCATACGA-3′) and (5′AATGATACGGCGACCACCGACAGGTT-3′) were used to generate DNA for sequencing. PCR was performed with an initial 5 min denaturation at 98 °C, followed by 10~14 cycles of 10 sec denaturation at 98 °C, 30 sec annealing at 54 °C and 15 sec extension at 72 °C. The PCR product was purified by run on a 6 % native polyacrylamide TBE gel and recovered by cutting the region of the gel between 100bp to 300bp. The product was purified through the gel extraction method. The prepared DNA was then sequenced on the Illumina Genome Analyzer II according to the manufacturer’s instructions with small RNA sequencing primer 5′-CGACAGGTTCAGAGTTCTACAGTCCGACGATC-3′.
characteristics.characteristic: description
characteristics.text: normal
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000461
characteristics.characteristic: disease
characteristics.text: Sendai virus cantell strain (ATCC VR-907)
characteristics.characteristic: infect
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: blood
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: GM0h
characteristics.characteristic: title