GSM9535989: RNA-seq 20201013_nih3t3_cas9_empty_vector_rep1; Mus musculus; RNA-Seq
Dynabeads M-280 sheep anti-Rabbit IgG (Invitrogen, 11201D) were diluted 1:10 in 1× PBS (Invitrogen, AM9624), magnetically washed, and incubated with Nesprin1 antibody (abcam, ab192234) at room temperature for 30 minutes. Antibody-coupled beads were washed and resuspended in cell lysis buffer containing 10 mM HEPES (Gibco, 15630106), 2.5 mM MgCl2 (Invitrogen, AM9530G), 10 mM KCl (Sigma, P9333), 1 mM DTT (ThermoFisher, 707265ML), 0.5% Triton X-100 (Sigma, 93443), and 2 U recombinant RNase inhibitor (Takara, 2313A). A population of 500 GFP-positive cells per sample was sorted into 10 µL lysis buffer in 96-well plates, centrifuged at 700 g for 1 minute, and incubated at 4°C with shaking (300 rpm) for 30 minutes. After magnetic separation, the supernatant was transferred to a new plate for RNA-seq workflow, while nuclei were retained for ATAC-side tagmentation workflow in AR-seq. RNA in the supernatant was purified using 2.2× VAHTS RNA Clean Beads (Vazyme, N412-03) and reverse-transcribed using SMART-seq2. RNA-seq library construction was performed using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501), following the manufacturer's manual. Libraries were generated from SMART-seq2-derived cDNA and sequenced in paired-end mode on short-read sequencing platforms.