title: GSM8881828: P12 Input Replicate A; Mus musculus; ChIP-Seq
library_construction_protocol: The samples were transferred to 1.5 ml tubes. SDS/RNase-free elution buffer (20 mM Tris–HCl/pH-7.5), 50 mM NaCl, 5 mM EDTA/pH-8.0) was added to 415 µl final volume, and the aqueous phase was sequentially extracted using equal volumes of phenol:chloroform:isoamylalcohol (Invitrogen, 15593-031) and chloroform:isoamylalcohol (Sigma-Aldrich, C0549). The 400 µl of the last aqueous phase were precipitated with 1 ml ice-cold ethanol, 11 µl linear acrylamide (Thermo Fisher Scientific, AM9520) and 44 µl 1 M sodium acetate (Invitrogen, AM9740) at least overnight at -80°C. For reconstitution, the pellets were centrifuged (swing-out, 16,000 x g, 4°C), and recovered using 15 µl of Qiagen EB (10 mM Tris, Qiagen, 19086) overnight at 4°C. Sequencing libraries were prepared using the Qiaseq ultralow input kit (Qiagen, 180492 + 180310) with modifications to minimize material loses. These included prolonged incubations during the AMPure XP bead extractions (>15 min bead/sample incubation, >15 min DNA elution) (Beckman coulter, A63881), and an increased volume of beads (1.2 volume equivalents) during the second bead extraction. Double-stranded DNA concentration was determined using the Qubit HS DNA kit (ThermoFisher Scientific, Q32851), while DNA fragment size analyses were performed with TapeStation D1000 HS (Agilent, 5067-5585). Libraries were then and pooled in equimolar amounts. Sequential AMPure XP extractions (1.2 volume equivalents) were employed to drastically reduce adapter dimers from the pool prior to sequencing.