title: GSM8296351: BM_256_Rep3_tHMGA2-GFP; Mus musculus; OTHER
library_construction_protocol: Genomic DNA (gDNA) from week 44 bone marrow cells were extracted using the Quick-DNA miniprep kit (Zymo Research). TRACE sequencing was performed as previously described [PMID: 35705805] with the following modifications. Tagmentation was done in an 80 µL reaction by incubating 500 ng of genomic DNA (gDNA) with 3 µL of Tn5 transposase (Diagenode; C01070010) and 4.8 µL of annealed Tn5 adapter top and bottom oligos at 45 µM in 5X TAPs buffer (50 mM TAPS-NaOH, pH 8.5, 25 mM MgCl2) with 8 µL DMF for 10 mins at 55°C. The reaction was quenched with 160 µL RLT buffer (Qiagen; 79216) followed by a 0.7X SPRI bead cleanup (Beckman Coulter; B23318). Tagmented DNA was eluted with 30 µL of nuclease-free water and 20 µL was used in the first round of PCR at 20 cycles with the Tn1c and LCI primers and Q5 High-Fidelity DNA Polymerase (NEB; M0494S), and the product was cleaned with 0.7X SPRI beads and eluted into 20 µL of nuclease-free water. 12 µL was then used for nested PCR at 20 cycles with primers MA-F and MA-LCII, and the product was purified with 0.7X SPRI-beads and eluted into 20 µL of nuclease-free water. 2 µL was used as input to indexing PCR with the NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) (NEB; E7600S) followed by a 0.7X SPRI bead cleanup. Final libraries were sequenced paired-end 150 x 2 (MiSeq Reagent Kit v2 300-cycles; Ms-102-2002) on an Illumina MiSeq.