title: GSM2396700: GRIDseq.mESC_rep1; Mus musculus; OTHER
library_construction_protocol: Approximately 2 million mammalian cells or 10 million Drosophila cells were sequentially crosslinked with 2 mM DSG and 3% formaldehyde. Nuclei were isolated, washed and digested in situ by AluI restriction enzyme. All DNA and RNAs in the nuclei were then treated by T4 PNK. After washing, nuclei were mixed with GRID-seq linkers and T4 RNA Ligase 2-truncated KQ for ssRNA ligation. Shorter strand of the linker was extended by SuperScript III Reverse Transcriptase to generate cDNA. Nuclei were washed again and resuspended in T4 DNA ligase solution for ligation of genomic DNA. By Proteinase K digestion at 65°C, linker-ligated DNA were reverse-crosslinked and extracted, followed by affinity purification by Streptavidin-conjugated beads. By denaturation with 150 mM NaOH solution, the non-biotinylated strand of the linker-ligated-DNA were released, and subjected to second strand synthesis by Klenow Fragment (3' to 5' exo-) enzyme. Newly synthesized dsDNA were then digested by MmeI for paired-end-tag generation, followed by size-selection in 12% native polyacrylamide gel. Recovered dsDNA was further ligated with adapters, PCR amplified to generate libraries for illumina sequencing.   GRO-seq protocol was adapted from (Core, L. J. et al, Science, 2008) Adapters were prepared by annealing the following two oligonucleotides in 1× NEB Buffer 2 to a final concentration of 25 mM: 5’-/5Phos/AGATCGGAAGAGCACACGTCT-3’ and 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN-3’. Purified DNA was dissolved in 10 µl of 1× NEB Buffer CutSmart and 0.5 U Shrimp Alkaline Phosphatase, incubated at 37 °C for 30 min and heat inactivated at 65°C for 5 min. The reaction was diluted with 36 µl H2O, mixed with 10 µl of 10× T4 DNA Ligase Buffer, 32 µl of PEG-6000, 200 pmol of Adapters and 1,600 U T4 DNA Ligase, and incubated at room temperature for 1 h. Unligated nick was phosphorylated by 20 U T4 Polynucleotide Kinase supplemented by 100 pmol ATP and incubated at 37°C for 30 min. Nick was then ligated by addition of 1 µl of 10× T4 DNA Ligase Buffer, 100 pmol ATP and 1,600 U T4 DNA Ligase, and incubation at room temperature for 30 min. DNA along with excessive Adapters were extracted and purified before loading to 10% native PAGE gel for size-selection. The desired band at approximately 185 bp was excised. DNA was extracted and dissolved in 20 µl of H2O. To amplify each library, 20 µl of PCR amplification mix (9.4 µl of H2O, 5 µl of DNA sample, 4 µl of 5× Phusion HF Buffer, 40 pmol dNTP, 5 pmol Primer#1, 5 pmol Primer#2, 0.4 U Phusion High-Fidelity DNA Polymerase was prepared. PCR primers consist of Primer#1 (5’- AATGATACGGCGACCACCGAGATCTACACBBBBBACACTCTTTCCCTACACGACGCTCTTCCGATCT -3’ (BBBBB: 5 nt barcode for multiplexing libraries)) and Primer#2 (5’- CAAGCAGAAGACGGCATACGAGACGTGTGCTCTTCCGATCT -3’).  PCR was performed with an initial 30 sec denaturation at 98°C, followed by 16 cycles of 10 sec denaturation at 98°C, 30 sec annealing at 65°C and 15 sec extension at 72°C. The PCR product was separated on a native 10% PAGE gel and the band at 188 bp was recovered. DNA was subsequently subjected to Illumina HiSeq 2500 for single-end 100 bp sequencing, with the sequencing primer (5’- ACACTCTTTCCCTACACGACGCTCTTCCGATCT -3’).
attributes.tag: GEO Accession
attributes.value: GSM2396700