GSM6831745: gDNA - Tracer mESC - Day3; Mus musculus; OTHER
gDNA was extracted from isolated Cas9-GFP+ Tracer mESCs at different days after Cas9-GFP activation for downstream intBCs library preparation. Briefly, 1x105-1x106 cell pellets from the indicated days were collected and lysed in 200µl Lysis buffer (10mM Tris-HCl pH=8.0; 10mM NaCl; 10mM EDTA; 0.5% SDS; 300 µg/mL Proteinase K) overnight at 55°C. The following day phenol:chloroform (Thermo, 15593031) extraction followed by precipitation overnight at -80°C (2.5V EtOH, 1/10V 5M NaCl and 1/100V molecular biology grade Glycogen (Thermo, R0561)) was performed to isolate gDNA. gDNA concentrations were measured using Qubit™ dsDNA HS- Kit (Thermo, Q32851) and same amount of input material was used for each sample for intBCs library preparation. Isolated gDNA from Tracer mESCs and prepared cDNA from Tracer mESCs and individual Tracer TLS were used for Amplicon-seq intBCs library preparation. 200ng gDNA and 100ng cDNA were used as input material. Two subsequent PCR reactions were carried on to prepare the NGS library: (i) in the first step the input material was used for 4x25µl PCR reactions for each sample. Amplification was carried out using Q5 2X MasterMix (NEB; M0492S) with the following steps: 98ºC 30sec/ 98ºC 10sec – 65ºC 30sec – 72ºC 45sec (12 cycles)/ 72ºC 5 minutes/ 4ºC hold. Next, the four reactions for each sample were pooled together and a double-sided AMPure XP beads (Beckman, A63881) cleanup (0.55x-0.8x) was performed and purified intBCs amplicons were eluted in 30µl EB buffer; (ii) in the second step, the whole eluate from the previous PCR reaction was used for 4x25µl PCR reactions for each sample. Amplification was carried out using Q5 2X MasterMix (NEB; M0492S) with the following steps: 98ºC 30sec/ 98ºC 10sec – 65ºC 30sec – 72ºC 45sec (12 cycles)/ 72ºC 5 minutes/ 4ºC hold. Next, the four reactions for each sample were pooled together and a AMPure XP beads (Beckman, A63881) cleanup (0.8x) was performed to purify amplicons libraries. Quality and concentration of the obtained libraries were measured using Agilent High Sensitivity D5000 ScreenTape on an Agilent 4150 TapeStation. Libraries were then sequenced using paired end sequencing (500 cycles kit; 250/250) on a Miseq platform at a minimum of 50 million fragments per sample.