| Library |
| Library name |
Construction protocol |
Strategy |
Source |
Selection |
Layout |
|
GRO-seq libraries were constructed as previously described with minor modifications (Wang et al, 2011). Briefly, nuclei were resuspended by nuclear run-on buffer and were incubated for 5 min at 30C in water bath, followed by proteinase K digestion and acidic phenol chloroform purification. After fragmentation, nascent RNAs with 5'-bromo-UTP were immuno-precipitated twice by using beads conjugated with an anti-BrdU antibody (Santa Cruz, sc-32323 AC). After poly-A tail addition and reverse transcription, the resulting first-strand cDNA was separated in a 10% TBE-urea polyacrylamide gel. cDNA that is 100-400 nt long was gel-recovered and self-ligated using CircLigase (Epicentre). The circularized single-stranded cDNA was relinearized with APE 1 (NEB) and then PCR-amplified using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) at optimal cycle numbers. The PCR products were separated in a 10% native TBE polyacrylamide gel and the 140-225 bp products were recovered. All libraries were sequenced on Illumina NovaSeq 6000 platform (SE 75 bp reads). |
OTHER |
TRANSCRIPTOMIC |
PCR |
SINGLE
|
|
