GSM3070787: 171213_CEMBA_mm_P56_P63_3C_MOp_CEMBA171207_3C_1_CEMBA171207_3C_2_A2_AD001_indexed; Mus musculus; OTHER
Bisulfite-converted samples were denatured at 95°C for 3 minutes, then placed on ice for 2 minutes. 5µL Random Priming master mix [1µL 10x Blue Buffer (Enzymatics B0110), 0.25µL Klenow Exo- (50U/µL, Enzymatics, P7010-HC-L), 0.5µL dNTP (10mM each, NEB N0447L), 3.25µL water] was added and incubated at 4°C for 5 min, 25°C for 5 min, and 37°C for 60 min followed by 4°C. 1.5µL enzyme mix containing Exonuclease 1 (20U/µL, Enzymatics X8010L) and rSAP (1U/µL, NEB M0371L) was added, then incubated at 37°C for 30 min followed by 4°C. 0.8x SPRI beads were added, mixed and incubated for 5 minutes at room temperature to allow DNA to bind. The beads were washed 3 times with 80% ethanol and eluted in 10µL EB buffer (Qiagen 19086). The plates were denatured again at 95°C for 3 minutes, and 10.5µL Adaptase master mix [2µL G1, 2µL G2, 1.25µL G3, 0.5µL G4 and 0.5µL G5 (Swift Biosciences 33096)] was added and incubated at 37°C for 30 minutes, then 95°C for 2 minutes. 25µL 2x KAPA HiFi HotStart ReadyMix (Kapa, KK2602) and 5µL custom indexing primer mix (6µM P5, 10µM P7) were added. The PCR was programmed as follows: 1) 95°C 2 min, 2) 98°C 30 sec, 3) 98°C 15 sec, 4) 64°C for 30 sec, 5) 72°C for 2 min, 6) 72°C 5 min, 7) 4°C hold. Repeat steps 3-5 for 15 total cycles. PCR reactions were cleaned with 0.8X SPRI beads for three rounds. Library concentration was determined with Qubit™ dsDNA BR Assay Kit (ThermoFisher Q32853). Libraries were sequenced using Illumina Novaseq instrument.
GEO Accession
GSM3070787