title: GSM4699998: Day0_Parental_1; Mus musculus; ATAC-seq
library_construction_protocol: Immediately following the nuclei prep, the pellet was resuspended in the transposase reaction mix (25 μL 2× TD buffer, 2.5 μL transposase (Illumina) and 22.5 μL nuclease-free water). The transposition reaction was carried out for 30 min at 37 °C. Directly following transposition the sample was purified using a Qiagen MinElute kit. Following purification, we amplified library fragments using 1× NEBnext PCR master mix and 1.25 μM of custom Nextera PCR primers 1 and 2 (Supplementary Table 1), using the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and thermocycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min. To reduce GC and size bias in our PCR, we monitored the PCR reaction using qPCR in order to stop amplification before saturation. To do this, we amplified the full libraries for five cycles, after which we took an aliquot of the PCR reaction and added 10 μl of the PCR cocktail with Sybr Green at a final concentration of 0.6×. We ran this reaction for 20 cycles to determine the additional number of cycles needed for the remaining 45-μL reaction. The libraries were purified using a Qiagen PCR cleanup kit yielding a final library concentration of ∼30 nM in 20 μL. Libraries were amplified for a total of 10–12 cycles.
attributes.tag: GEO Accession
attributes.value: GSM4699998