GSM9350242: Female, KO, rep6; Mus musculus; RNA-Seq
Total RNA was extracted using Trizol, and cleaned up with a RNeasy Mini Kit (Qiagen). Messenger RNA was purified from 700ng-1000ng total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). First-strand cDNA was synthesized from heat-denatured purified mRNA using a Maxima H Minus First Strand cDNA Synthesis kit (Thermo-Fisher) and random hexamer primers, followed by PCR Clean DX (Aline Biosciences) bead purification on a Microlab NIMBUS robot (Hamilton Robotics). The second strand cDNA was synthesized following the NEBNext Ultra Directional Second Strand cDNA Synthesis protocol (NEB) with dUTP incorporation. cDNA fragmented to 250-300bp using Covaris LE220, followed by end-repair, A-tailing, and Illumina adapter ligation. Products digested with USER enzyme and immediately amplified with 10 cycles of indexed PCR using NEBNext Ultra II Q5 polymerase (NEB) and Illumina's dual index primer set. Final libraries purified, size-selected with PCR Clean DX beads, and QC performed using Agilent DNA 1000 and Qubit quantification before pooling for paired-end 150 bp sequencing.