shRNA-mediated knockdown in primary cells

HEK293T/17 cells (ATCC, CRL-11268) were used to produce virus particles containing shRNA sequence. The HEK293T/17 cells were grown in DMEM media (Thermo Fisher, 11965092) with 10% FBS (Fisher Scientific, 10437028) were transfected with lentiviral pLKO.1 plasmid construct with shRNA against human HOXA13 (Sigma, TRCN0000015406) along with pMD2.G and psPAX2 plasmids. Transfection was performed using Lipofectamine 2000 reagent (ThermoFisher, 11668019) according to manufacturer’s instructions. Media was changed after overnight incubation. Virus particles were collected 24 hours later by spinning down the supernatant and stored at −80°C in small aliquots when not used immediately. 2 × 10⁵ primary leiomyoma cells (Passage 2 or 3) were transduced in a 6-well plate with either control or HOXA13 gene silencing lentiviral particles in the presence of 6ug/ml polybrene for 18 hours. Fresh SmGM complete media was added and then after 24 hours, cells were selected in 2ug/ml puromycin for 3 days. Cells were then harvested for RNA using RNeasy mini kit (QIAGEN, 74104) and for protein with modified RIPA lysis buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% IGEPAL CA-630, 1% sodium deoxycholate, 0.25% SDS). Whole-cell extracts prepared using modified radioimmunoprecipitation assay (RIPA) buffer were processed as described previously (Parker et al., 2012). Extracts were clarified by centrifugation at 20,000 × g for 15 min at 4°C, and protein concentrations determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, 23225). About 30μg of protein lysate was loaded on precast 8 to 16% polyacrylamide gels (Thermo Fisher Scientific, XP08162BOX). Following transfer to nitrocellulose membrane, western blot was performed to detect HOXA13 (Abcam, ab106503). Anti- GAPDH (Sigma-Aldrich, G9545) antibody was used as a loading control. About 100ng of RNA was used for library preparation using TruSeq stranded mRNA kit (Illumina, 20020594) and paired-end sequenced on Illumina Nextseq 500/550 as described above.

In Figure S7C and S7D, protein and RNA were extracted from the primary leiomyoma cells 5 days after being transduced with lentivirus containing negative control (shControl) or HOXA13 (shHOXA13) shRNA construct.