HumanNeuronCulturePositiveSingle, rep4
We designed a novel library protocol named CHEX-seq (CHromatin EXposed), which couples oligonucleotide random annealing and nucleus-resolution photoactivation such that the single-stranded open-chromatin DNA is copied, reverse transcribed, amplified and sequenced. Specifically, the protocol involves four major steps. (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 degree Celsius after priming with custom App-RC-polyC primer. 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 and 2nd strand DNA was synthesized using DNA Polymerase 1 with a custom XXbpPBCYY primer (XX=16-18, YY=1, 15-24). Subsequently, the double-stranded blunt ended DNA was amplified using custom primers XXbpPBCYY / App-RC following PCR condition: 98 degree Celsius for 30 sec; thermocycling at 98 degree Celsius for 10 sec, 50 degree Celsius for 30 sec, 72 degree Celsius for 30 sec for 27 cycles; extension at 72 degree Celsius for 2 mins and was then used for library construction. To distinguish CHEX-seq priming from endogenous PCR priming, the PCR primers were designed with their 3 ends clipped up to 4bp. 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. (D) Sequencing library preparation: Illumina TruSeq Nano DNA Library Preparation Kit was used with modifications. The entire 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.