GSM7298774: K562PositiveSingle, rep6; Homo sapiens; OTHER
After fixation and permeabilization, the cells and brain slices were incubated with CHEX-seq probe (170 nM) in TES buffer (10 mM Tris, 1 mM EDTA, 150 mM NaCl) for 1 hr at room temperature. The samples were then washed with 1x PBS (w/o Ca++, Mg++) 3 times for 3 min. After CHEX-seq probe annealing and washing, the samples were transferred to the imaging chamber with 1x PBS (w/o Ca++, Mg++). All images and photoactivations were performed using a Carl Zeiss 710 Meta confocal microscope (20x water-immersion objectives, NA 1.0). CHEX-seq probe annealing was confirmed by exciting at 633 nm and emission was detected at 640-747 nm. The photoactivation was performed using the 405 nm (UV) laser at 60% power and 6.30 μs per pixel. After photoactivation in each individual cell's nucleus, a master mix containing DNA polymerase I and 1st strand DNA synthesis buffer was added to the cells and incubated for 1 h at room temperature. Subsequently, the single cells containing synthesized complementary DNA were harvested using a glass micropipette under using a Zeiss 710 confocal microscope (Carl Zeiss) for visualization. (A) 1st strand DNA synthesis and poly G tailing at 3' end: After harvesting single cells, the in situ synthesized cDNA was removed by adding fresh prepared 0.1 N NaOH and incubating the sample for 5 min at RT followed by neutralization with 1 M Tris (pH 7.5). After ethanol precipitation, the 1st strand DNA was resuspended in nuclease free water. Subsequently, poly(G) was added to the 3' end using terminal deoxynucleotidyl transferase (TdT) (Invitrogen). (B) 2nd strand DNA synthesis and round 1 linear RNA amplification: 2nd strand DNA was synthesized using DNA polymerase I for 2 h at 16 °C after priming with custom App-RC-polyC primer (Supplemental Data, Table 1). RNA was amplified using linear in vitro transcription from T7 RNA polymerase promoter incorporated into the double-stranded DNA with Ambion MEGAscript T7 In Vitro Transcription (IVT) Kit. (C) Round 2 1st and 2nd strand DNA synthesis and PCR amplification: After cleanup IVT reaction, 1st strand DNA was reverse transcribed from aRNA using Superscript III using a custom App-RC primer (Supplemental Data, Table 1) 2nd strand DNA was synthesized using DNA Polymerase 1 with a custom 18bpPBC1 primer (Supplemental Data, Table 1). Subsequently, the double-stranded blunt ended DNA was amplified using custom primers 18bpPBC1 / App-RC (Supplemental Data, Table 1) following PCR condition: 98 °C for 30 sec; thermocycling at 98 °C for 10 sec, 50 °C for 30 sec, 72 °C for 30 sec for 27 cycles; extension at 72 °C for 2 mins, and was then used for library construction. Samples for the control experiments were processed with the same procedure except no CHEX-seq probe was applied, and 2nd round 2nd strand DNA PCR amplification was performed with custom primers 18bpPBC14 / App-RC. Illumina TruSeq Nano DNA Library Preparation Kit was used with modifications. All of the second round PCR amplified double-stranded DNA was used as input. After converting DNA fragment into blunt ends with End Repair Mix, base “A” was added; sequence adapters were ligated. DNA inserts were amplified with PCR.