GSM8647845: parental_IVT_rep1; Mus musculus; OTHER
Qiagen DNA mini kit The same protocol was followed both for shuffle cassette insertion site mapping from the parental population and for rearrangement call validation post Cre transfection. Genomic DNA purified using the Qiagen DNeasy kit was used as template for all IVT reactions. For loxPsym+ mESC samples, 300 ng of template, for loxPsym+ K562, attB/P+ mESC and attB/P+ K562, 500ng of template was used per reaction. T7 IVT was performed using the HiScribe T7 High Yield RNA Synthesis Kit (NEB E2040S), in a 60 μl total reaction for loxPsym+ mESCs and in a 30 μl total reaction for all other samples. Reactions were incubated at 37°C for 16 hours in a thermocycler with the lid set to 50°C. Reactions were treated with Turbo DNase (Thermo AM2238) to remove template DNA according to manufacturer's instructions and RNA was extracted using Trizol LS reagent (Thermo 10296010). Briefly, each sample volume was normalized to 250 μl with water, 750 μl of Trizol LS reagent was added. Samples were mixed by pipetting and incubated at room temperature for 4 min. 200 μl of chloroform was added and samples were incubated at room temperature for 3 min. Samples were then spun at 12,000 x g for 15 min and the aqueous phase was transferred to a new tube. 1 μl of 5 mg/mL Glycogen was added (Invitrogen) per sample. Next, RNA was precipitated by adding 1 volume of isopropanol. Samples were mixed by inverting and incubated at -80°C for 1 hour and subsequently spun at 21,000 x g for 1 hour at 4°C. RNA pellet was washed with ice cold 80% ethanol and resuspended in 11.5 μl of H2O. Reverse transcription was performed using the SuperScript IV Reverse Transcriptase (Thermo 18090200) with 0.5 uL 100 μM RT primer that contains an 8 base-pair degenerate 3' end (oSP1012). RNA was initially incubated with 0.5 μL 100 μM RT primer (oSP1012) and 1 μL 10 mM dNTP at 65°C for 5 min and cooled on ice. Enzyme, buffer, RNAse inhibitor and DTT were added and reactions were incubated in a thermocycler at 23°C for 10 min, 50°C for 15 min and 80°C for 10 min, followed by a hold at 10°C.  For loxPsym+ mESC replicate 1, 2nd strand synthesis for both top and bottom strands was performed in the same reaction with primers oSP1008, oSP1021 and oSP1013. For loxPsym+ mESC replicate 2, 2nd strand synthesis for top and bottom strands were performed separately, one with oSP1008 and oSP1013 and another with oSP1021 and oSP1013. Four 50 μl PCR reactions were performed per sample with Q5 Polymerase (NEB M0492S) with the following cycling parameters: 98°C - 3 min; 4 cycles of 98°C - 20s, 65°C - 20s, 72°C - 30s; 72°C - 60s, hold at 4°C. Reactions corresponding to a particular sample and primer pair were pooled. For replicate 1, double-sided size selection (0.5X, 1.1X) was performed with Ampure XP beads (Beckman A63882) on 200 μl of sample and eluted in 50 μl of H2O. For replicate 2, double-sided size selection (0.5X, 1.1X) was performed on 100 μl of sample and eluted in 25 μl of H2O. From each sample, a second PCR was set up with indexing primers using 12 μl of the previous eluate as input. Two 50 μl PCR reactions were performed per sample with Q5 Polymerase (NEB M0492S) with real-time tracking using SYBR green dye with the following cycling parameters 98°C - 3 min; 4 cycles of 98°C - 15s, 65°C - 15s, 72°C - 30s until the curves reached saturation (14-16 cycles). Running these libraries on a D1000 ScreenTape (Agilent) revealed a smear of expected size but also some lower molecular weight products that may dominate the sequencing reaction. To address this, we created 3 equimolar pools: samples from replicate 1, top strand samples from replicate 2 and bottom strand samples from replicate 2. Pools were run on a 6% TBE PAGE gel and DNA between 400 bp and 1000 bp was excised and purified.  For both replicates of loxPsym+ K562, attB/P+ mESC and attB/P+ K562 samples, 2nd strand synthesis was performed according to the replicate 2 protocol described above with minor modifications. Two 50 μl PCR 1 reactions were performed per sample, one corresponding to the top strand and one for the bottom strand. After bead clean-up, one 50 μl PCR 2 reaction was performed per sample, per strand. PCR2 was performed with the previous cycling parameters for a total of 16-18 cycles. Libraries were run on agarose gel and DNA between 400 bp and 1000 bp was excised and purified.  Library sequencing was performed on an Illumina NextSeq2000 P2 300 cycle kit. For loxPsym+ mESCs, the read lengths were: 106 read1, 10 index1, 10 index2 and 212 on read2. For all other samples, read lengths were: 124 read1, 6 index1, 10 index2 and 198 on read2.  Primer sequences:  oSP1012_IST_RT_TruSeqR2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNN oSP1013_TruSeq_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT oSP1008_996_CS2_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNCCTTAGCCGCTAATAGGTGAGC oSP1021_1014_CS1_Nextera1_10N_F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNTTGCTAGGACCGGCCTTAAAGC