title: GSM1480322: K562 GRO-cap control; Homo sapiens; OTHER
library_construction_protocol: Nuclei isolation: All steps performed and buffers kept at 4oC. 10 mls of cells were spun down at 700g in a swinging bucket rotor for 5 min. Cells were washed in 1X cold PBS. Cells were washed in 10ml of buffer W (10mM Tris-Cl, pH 7.5, 10mM KCl, 150mM sucrose, 5mM MgCl2, 0.5mM CaCl2, and 0.5 mM DTT). Cells were resuspended in 5mls of Buffer P (10mM Tris-Cl, pH 7.5, 10mM KCl, 250mM sucrose, 5mM MgCl2, 1mM EGTA, and 0.5 mM DTT, .05% tween-20 and/or .1% Igepal), and incubated 5 min. on ice. Permeabilized cells were washed twice in 5ml of buffer P, resuspended in 1ml of buffer P, and transferred to a 1.7ml eppendorf tube. Cells were pelleted at 1000 x g in a fixed angle rotor for 5min and then resuspended in 500 ml of buffer F (50 mM Tris-Cl pH 8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA, 0.5mM DTT). Cells were pelleted at 100g for 5 min, resuspended in 100 ml of buffer F, and then flash frozen in liquid nitrogen. All NRO-reactions and bead enrichment steps for GRO-cap were carried out as described in GRO-seq, with the exception that the RNA for GRO-cap was not base hydrolyzed. After the first bead binding, the TruSeq RNA 3' Adapter (RA3) (Illumina part # 15013207) was ligated to the 3'-end of the NRO RNA. First, 50 pmoles of the 3'-adapter were mixed with NRO RNA, and 2 ml 50% PEG 8000, brought to 14 ml with DEPC water, incubated at 70°C for 3 min and put on ice for 2 min. Then, 2 ml of 10X T4 RNA Ligase I buffer, 2 ml 10 mM ATP, 1 ml SUPERaseIN, and 1.5 ml T4 RNA Ligase I (NEB, M0204) were added and reaction incubated at 22°C for 4-6 hr. The reaction was then brought to 100 ml with binding buffer and subjected to a second round of bead enrichment. After the second bead enrichment, 5' mono-phosphate RNAs were selected against in two successive steps. First, to selectively degrade RNAs with a 5'-mono-phosphate, NRO RNA was resuspended in 16.5 ml DEPC water, mixed with 0.5 ml SUPERaseIN, 2 ml 10X Terminator reaction buffer A, and 1 ml of Terminator 5'-phosphate-dependent exonuclease (Epicentre, TER51020), and incubated at 30°C for 1 hr. The reaction was extracted and precipitated using the standard method (above), and resupended in 10 ml DEPC water. Second, 5'-mono-phospate RNAs were deposphorylated to prevent their participation in subsequent ligation reactions. For this, the RNA was then mixed with 1 ml SUPERaseIN, 14.5 ml DEPC water, 10X Antarctic Phosphatase buffer, 1.5 ml of Antarctic Phosphatase (NEB, M0289S), and incubated at 37°C for 30 min. The reaction was brought to 200 ml with 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, and heat inactivated at 65°C for 5 min. The reaction was then extracted and precipitated using the standard extraction method and resuspended in 10 ml DEPC water. The 5'-capped RNAs then were prepared for ligation and the final library preparation steps. The NRO RNAs were then split in half and the experimental sample was treated with Tobacco Acid Pyrophosphatase (TAP): 1 ml SUPERaseIN, 15 ml DEPC water, 3 ml 10X TAP buffer, and 1 ml TAP (Epicentre, T19500), incubation at 37°C for 1 hr. The control reaction was treated identically except for the addition of TAP. The reaction was brought to 200 ml and then extracted and precipitated using the standard method. The TruSeq RNA 5' Adapter (RA5), (Illumina part # 15013205) was ligated to the 5'-end of the NRO RNA. First, 50 pmoles of the 5'-adapter were mixed with NRO RNA, and 2 ml 50% PEG 8000, brought to 14 ml with DEPC water, incubated at 70°C for 3 min and put on ice for 2 min. Then, 2 ml of 10X T4 RNA Ligase I buffer, 2 ml 10 mM ATP, 1 ml SUPERaseIN, and 1.5 ml T4 RNA Ligase I (NEB, M0204) were added and reaction incubated at 22°C for 4-6 hr. The reaction was then brought to 100 ml with binding buffer and subjected to a third round of bead enrichment. After the third enrichment, samples were reverse transcribed, amplified and PAGE purified and quantified before submission for sequencing.
attributes.tag: GEO Accession
attributes.value: GSM1480322