title: Illumina HiSeq 4000 paired end sequencing; Identifying LINC00899 binding sites in HeLa cells with CHART-seq
design: Identifying LINC00899 binding sites in HeLa cells with CHART-seq
library_construction_protocol: Cells were collected at 70-80% confluence. Five 150 mm dishes were used for each pulldown. Cells were trypsinized, washed with phosphate buffer saline solution, and fixed twice with 1% formaldehyde. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 degrees Celsius in 5% CO2. Nuclei were extracted and cross-linked with 3% formaldehyde for 30 minutes at room temperature. Nuclei were washed 5 times after quenching with 0.125 M glycine. Sonication was performed using a Covaris S220. Extracts were collected and hybridised to capture oligonucleotides (The antisense pool was comprised of 4 separate oligonucleotides with sequences derived from the antisense strand of LINC00899, the antisense pool, derived from the sense strand of LINC00899, was a mix of 2 separate oligonucleotides, which were the direct reverse complement sequences of two of the oligos in the antisense pool) at a final concentration of 25 uM, overnight at room temperature. A portion of the nuclear extract incubated with either the sense or antisense capture oligos was removed prior to pulldown to serve as input controls. Streptavidin C1 beads were used to capture complexes overnight at room temperature. Bound material was washed 5 times and RNAse H was added for 30 minutes at room temperature to elute RNA-chromatin complexes. Eluates were reverse-cross-linked and treated with Proteinase K (55 degrees Celsius for 1 hour, then 65 degrees Celsius for 30 minutes). DNA was purified using phenol-chloroform extraction and ethanol precipitation. Final CHART DNA was eluted in 12.5 uL of Tris-EDTA buffer. 10 uL from this reaction was used for sonication using the Covaris LE220 to obtain fragment lengths below 500 bp. CHART DNA  was used for library preparation using the ThruPLEX DNA-seq library preparation protocol (Rubicon Genomics). For inputs, 6 PCR cycles were used, while for pulldown eluates, 8 PCR cycles were used. Library fragment sizes were performed using a 2100 Bioanalyzer and libraries quantified by the KAPA quantification kit.
attributes.tag: Experimental Factor: pull-down
attributes.value: input (no pull-down)
attributes.tag: Experimental Factor: capture oligonucleotide
attributes.value: pool of 2 lncRNA LINC00899_sense oligonucleotides