title: GSM1548734: MspI_reference_Kdm5b_KD; Mus musculus; OTHER
library_construction_protocol: mES cells were collected by washing and trypsinizing for three minutes, and FACS sorted by the GFP signal present in the transfected plasmid. Genomic DNA was extracted by resuspending the cells in lysis buffer (10 mM TE, 150 mM EDTA and 1% SDS) supplemented with 10 mg/ml of RNase A and 20 mg/ml of Proteinase K, and incubated at 50 °C overnight.  The lysed cells were phenol-chloroform extracted and the resultant DNA was dialyzed in 0.2X SSC buffer (300 mM NaCl and 3 mM Na3C6H5O7, ph 7.0) for 24 hours.  The DNA sample was concentrated in the dialysis bags using polyethylene glycol, following which the quality and concentration of the DNA were measured by Nanodrop spectrophotometry. Two custom adapters were created for HELP-tagging, referred to as AE and AS.  As well as an Illumina adapter sequence, adapter AE contains an EcoP15I recognition site and a T7 promoter sequence.  Adapter AS contains an Illumina sequencing primer sequence.  One micrograms of genomic DNA were digested with HpaII and MspI in separate 50 μl reactions and purified by phenol/chloroform extraction followed by ethanol precipitation.  The digested genomic DNA was ligated to adapter AE using an NEB Quick Ligation Kit (25 μl of 2x Quick ligase buffer, 1 μg of MspI/HpaII-digested DNA , 0.1 μl of Adapter AE (1 μM), 3 μl of Quick Ligase in a final volume of 50 μl).  After AE ligation, the products were purified using AMpure (Agencort), then digested with EcoP15I (NEB).  The restriction fragments were end-repaired to inhibit dimerization of adapters, and tailed with a single dA, at the 3’ end.  After the dA tailing reaction, adapter AS was ligated to the dA-tailed fragments using an NEB Quick Ligation Kit (25 μl of 2x Quick ligase buffer, 2.5 μl of Adapter AS (10 μM), 2.5 μl of Quick Ligase in a final volume 50 μl).  After ligation, products were purified using the MinElute PCR purification kit (Qiagen) and in vitro-transcribed using MEGAshortscript (Ambion).  Following in vitro transcription, products were purified with an RNeasy clean-up kit (Qiagen) before reverse transcription was performed using the SuperScript III kit (Invitrogen).  The first strand cDNA produced was used as a template for PCR using the following conditions: 98°C for 2 minutes, then 18 cycles of 98°C for 15 seconds, 60°C for 15 seconds and 72°C for 15 seconds followed by 5 minutes at 72°C for the final extension.  The product then underwent gel extraction (Qiagen).