title: GSM3307381: siNXF1_rep2_3READ; Homo sapiens; RNA-Seq
library_construction_protocol: See the "extraction protocol" column in the SAMPLES section. 3'READS: The 3'READS+ version was used for 3' end sequencing, as previously described (Zheng et al., 2016).  Briefly, RNA samples (total or nuclear or cytoplasmic RNA) were prepared from HeLa cells were treated with Cntl siRNA, ALYREF siRNA, THOC2 siRNA and CFI-68 siRNA.   Poly(A)+ RNA in 10 μg of total RNA was captured using oligo (dT)25 magnetic beads (NEB) in binding buffer (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 0.05% TWEEN 20) and fragmented on the beads using RNase III (NEB) at 370C for 15 min.  After washing with binding buffer, poly(A)+ fragments were eluted from the beads with TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.5) and precipitated with ethanol, followed by ligation to heat-denatured 5'adapter (5′-CCUUGGCACCCGAGAAUUCCANNNN, Sigma).  The ligation products were captured by biotin-T15-(+TT)5 attached to Dynabeads MyOne Streptavidin C1 (Life Technologies).  After washing with washing buffer (10 mM Tris-Cl, pH 7.5, 1 mM NaCl, 1 mM EDTA, and 0.05% TWEEN 20), RNA fragments on the beads were incubated RNase H (Epicentre) at 370C for 30 min. Following washing with RNase H buffer, RNA fragments were eluted from the beads in elution buffer (1 mM NaCl, 1 mM EDTA, and 0.05% TWEEN 20) at 500C, precipitated with ethanol, and then ligated to heat-denatured 5' adenylated 3' adapter (5'-rApp/NNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC).  The ligation products were then precipitated and reverse transcribed using M-MLV reverse transcriptase (Promega), followed by PCR amplification using Phusion high-fidelity DNA polymerase (NEB) and bar-coded PCR primers for 12-18 cycles.  PCR products were size-selected twice with AMPure XP beads (Beckman Coulter), using 0.6 volumes of beads (relative to the PCR reaction volume) to remove large DNA molecules and an additional 0.4 volumes of beads to remove small DNA molecules. The eluted DNA was selected again with 1 volume of AMPure XP beads to further remove small DNA molecules.  The size and quantity of the libraries eluted from the AMPure beads were examined using a high sensitivity DNA kit on an Agilent Bioanalyzer (Agilent).  The library concentrations were further measured by qPCR using primers corresponding to 5' and 3'end regions of cDNAs. Libraries were sequenced on an Illumina HiSeq 2000 machine (1 × 50 bases).                                                           RNA-seq of nascent RNA: Nascent RNA sequencing was largely adapted from the NET-seq (Mayer et al., 2015) and TT-seq methods (Schwalb et al., 2016), with some modifications.  For each sample, cells at 75~90% confluency on a 15-cm dish were treated with 500 µM of 4sU for 5 min before harvest.  Cells were washed with ice-cold PBS buffer, and scraped off from the dish in 10 ml of ice-cold PBS while on ice.  After centrifugation at 420 x g at 40C for 2 min, cells were lyzed by incubation with 4 ml of ice-cold HLB+N buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2 and 0.5% (vol/vol) NP-40) on ice for 5 min.  1 ml of ice-cold HLB+NS buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2, 0.5% (vol/vol) NP-40 and 10% (wt/vol) sucrose) was underlaid to the cell lysate and the lysate was centrifuged at 420x g at 4 °C for 5 min. After removal of the supernatant, the pelleted nuclei were re-suspended in 125 μl of ice-cold NUN1 buffer (20 mM Tris-HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA and 50% (vol/vol) glycerol) by pipetting up and down, mixed with 1.2 ml of ice-cold NUN2 buffer (20 mM HEPES-KOH (pH 7.6), 300 mM NaCl, 0.2 mM EDTA, 7.5 mM MgCl2, 1% (vol/vol) NP-40 and 1 M urea) by pulse vortexing, and incubated on ice for 5 min. The mixture was centrifuged at 14,000x g at 4 °C for 2 min to pellet chromatin. After removal of the supernatant completely, RNA was extracted from the chromatin pellet using 1 ml TRIzol.       Extracted chromatin RNA from each sample (4 µg) was fragmented at 94 °C for 1 min using NEBNext Magnesium RNA Fragmentation Module (NEB). The reaction was then diluted to 100 µl with nuclease-free H2O, mixed with 5 µg of glycogen, 10 µl of 3M Sodium Acetate (pH 5.5), and 300 µl of ethanol. After precipitation at -80 °C for 45 min, the RNA was pelleted by centrifugation at 14,000x g at 4 °C for 20 min. The pellet was washed with cold 75% ethanol, air-dried, and dissolved in 5 μl of 2 µM of 5' adenylated 3' blocked 3' adapter (5'-Aden/NNNNNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC, where N is a random nucleotide). After incubation at 70 °C for 2 min, the sample was immediately chilled on ice, and incubated with ligation mix at 25°C for 2 h. The ligation mix contained 2 µl of nuclease-free H2O, 6 µl of 50% PEG 8000, 1.5 µl of 10x T4 RNA ligase buffer, 0.25 µl of SuperaseIn (20 U/µl), and 0.25 µl of truncated RNA ligase 2 (KQ, 200 U/µl).  To biotinylate 4sU-labeled RNA fragments, the ligation reaction was mixed with 125 µl of nuclease-free H2O, 20 µl of 10x biotinylation buffer (100 mM Tris pH 7.4, 10 mM EDTA), and 40 µl of biotin-HPDP (1 µg/µl in DMF).  The mixture was incubated at room temperature on a rotator for 1.5 hr.  The reaction was extracted with 200 µl of chloroform three times and the RNA in the aqueous phase was precipitated as described above.      Biotinylated RNA pellet was dissolved in 50 µl of nuclease-free H2O and denatured by incubation at 700C for 2 min.  After chilling on ice, the RNA was mixed with 50 µl of Streptavidin C1 Dynabeads in 2x B&W buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 2 M NaCl) supplemented with 0.5 µl of SuperaseIn (20U/µl).  The mixture was incubated on a rotator at room temperature for 15 min.  After removal of the supernatant on a magnetic stand, the beads were washed three times with 0.5 ml of 1x B&W buffer (5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl) at 65 °C, followed by three washes with 1x B&W buffer at room temperature.  After complete removal of the washing buffer, the beads were re-suspended with 30 µl of the PNK reaction mix, which contained 3 µl of 10X PNK buffer, 22 µl of H2O, 3 µl of 10 mM ATP, 1.5 µl of PNK (10U/µl), and 0.5 µl of SuperaseIn (20U/µl).  This step removes the 5' phosphate group from RNA.  The mixture was rotated in an oven at 370C for 30 min and then mixed with 170 µl of 1x B&W Buffer containing 100 mM DTT and incubated at room temperature for 3 min to elute 4sU-labled RNA.  After collecting the beads on a magnetic stand, the supernatant was transferred to a new tube. The beads were subjected to another round of RNA elution using 200 µl of 1x B&W Buffer containing 100 mM DTT. The eluted RNA was precipitated as described before.     Isolated 4sU-labeled RNA was then subject to 5' adapter ligation, reverse transcription and cDNA library construction. First, the RNA was dissolved in 5 µl of H2O and mixed with 1 µl of 5 µM 5' RNA adapter (5'-CCUUGGCACCCGAGAAUUCCA). After incubation at 700C for 2 min, the mixture was chilled on ice and mixed with 0.8 µl of H2O, 1 µl of 10 mM ATP, 1 µl of 10x T4 RNA ligase buffer, 0.2 µl of SuperaseIn (20U/µl), and 1 µl of RNA ligase 1 (20U/µl). The reaction was incubated at 25 °C for 2 h. The ligation product was mixed with 1.5 µl of 5 µM RT primer (5'-GTTCAGAGTTCTACAGTCCGACGATC) and reverse transcribed in a 15 µl reaction following Promega's M-MLV reverse transcription protocol. cDNAs were amplified by 18 cycles of PCR using Phusion DNA polymerase in a 50 µl reaction containing an indexed PCR primer and primer GX3 (5'-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA). PCR products were size-selected, quality-checked, and sequenced as described above.                                                        ChIP-seq: HeLa cells transfected with control or NXF1 siRNA were crosslinked with 1% for formaldehyde for 10 min at room temperature.  Crosslinks were quenched in 125 mM glycine for 5 min at room temperature, and cells were washed with cold PBS and then collected by centrifugatio
attributes.tag: GEO Accession
attributes.value: GSM3307381