GSM1666528: KBM7 cell line, clone 5-5, haploid, sample 133; Homo sapiens; OTHER
Based on the FCS and SSC profiles the 2% smallest cells that were double positive for the Fucci markers were single cell sorted in a PCR plate containing lysis buffer and proteinase K (Costar Thermowell). Each well contained 1μl of pick buffer (50mM Tris-HCl pH 8.3, 75mM KCl, 3mM MgCl2, 137mM NaCl) and 2μl of lysis buffer with proteinase K (10mM Tris acetate pH 7.5 (Sigma T1258), 10mM magnesium acetate (Sigma 63052), 50mM potassium acetate (Sigma 95843), 0.67% Tween-20 (Sigma P2287), 0.67% Igepal (Sigma I8896), 0.67mg/ml proteinase K (Roche 03115828001). Proteinase K digestion was performed at 42 °C for 4 hours in a thermoblock with heated lid, followed by heat inactivation for 10 minutes at 80 °C. In the following steps, reagents were added with an Eppendorf Mulipipette plus mounted with a 0.1 ml Combitip. Genomic DNA (gDNA) was digested for 4 hours by the addition of DpnI (1 unit) in 7μl 1* One-Phor-all-buffer plus (10mM Tris acetate, pH7.5, 10mM magnesium acetate, 50mM potassium acetate) at 37 °C, followed by heat inactivation at 80 °C for 20 minutes. Adaptor ligation was performed by the addition of 10μl ligation mix (2* Roche T4 ligation buffer, 0.5 units T4 ligase, 50μM double-stranded DamID adapter {Vogel, 2007, 17545983}) and incubated at 16 °C over night. Heat inactivation at 65 °C for 10 minutes the next day was followed by PCR amplification by the addition of 30 μl PCR mix (5 μl 10* Clontech Advantage cDNA reaction buffer, 4 μl dNTPs (2.5mM each), 1.25 μl primer (50 μM), 1 μl PCR advantage enzyme mix (50*) and 18.75 μl H2O).  The PCR reaction was performed with 21 extension steps as described in Vogel et al., 2008, Nature Protocols. The PCR primer consists of an additional 4 random nucleotides at the 5' end to meet the Illumina software requirements of generating reads with diverse starting sequences. Of the PCR material 8μL was submitted to standard 1% agarose gel electrophoresis for analytical purpose and the successfully amplified material was purified by column purification (Qiagen PCR purification kit). Next the samples were prepared for Illumina sequencing. Of 1μg purified PCR product the 3’ or 5’ overhanging ends were blunted in a 50μl reaction following the manufacturers intructions (End-It DNA End-Repair Kit, Epicentre). Next, a 3’ adenine was added by incubation of the DNA at 37°C in a 50 μL reaction mix (1* New Engeland Biolabs restriction buffer 2, 200μM dATP, nuclease-free water) containing klenow 3′ → 5′ exo– (New Engeland Biolabs, 100 units final concentration). After incubating the samples for 45 minutes, the DNA was purified with 1.6X Agencourt AMPure XP beads (Beckman Coulter). To 250 μg of purified DNA Illumina Y-shaped adapters were ligated for two hours at room temperature in a 10μl reaction mix (1* Roche ligation buffer, 0.25 units T4 DNA (Roche 5units/μl, 50μM Y-adapter, nuclease-free water). Next, the T4 ligase reaction was heat inactivated at 65 °C for 10 minutes followed by DNA purification as described for the previous step. For the addition of the Illumina index primers a PCR reaction was performed with 100 ng DNA from the previous step in a 20 μl MyTaq red DNA polymerase (Bioline BIO21110) PCR reaction mixture (10μl 1* MyTaq reaction mixture, 0.25 μM Illumina P5 primer, 0.25 μM Illumina indexing primer, nuclease-free H2O). The DNA was amplified for 8 PCR amplification cycles (94 °C 1 minute, 94 °C 30 seconds, 58 °C 30 seconds and 72 °C for 30 seconds) after which 5 μl of each sample was analysed by agarose gel electrophoresis. For Illumina multiplex sequencing typically between 40 and 50 samples were mixed in equimolar ratios based on the agarose gel image.
GEO Accession
GSM1666528