GSM6831753: cDNA - Tracer mESC - Day3; Mus musculus; OTHER
RNA was extracted from isolated Cas9-GFP+ Tracer mESCs at different days after Cas9-GFP activation or from individual Tracer TLS at day 5 post aggregation for downstream intBCs library preparation. Briefly, RNA was extracted from 1x105 isolated Cas9-GFP+ Tracer mESCs or individual Tracer TLS using RNeasy Plus Micro Kit (Qiagen) and RNA concentration and quality was measured using Qubit™ RNA HS (Thermo, Q32852). cDNA synthesis was performed using 1μg total RNA (for Tracer mESCs) or all extraced RNA (for individual Tracer TLS) using the RevertAid First Strand cDNA Synthesis Kit (Thermo, K1622), following the manufacturer's instructions. OligoT were used as primers first strand cDNA synthesis. 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.