Plko 1 puro

The lentiviral transduction of shRNA targeting CHRDL1 (shCHRDL1) or mammalian non-targeting Control-shRNA (shCtrl) was performed as described previously [29 (link),30 (link)] after the transfection of the shRNA-containing vector (pLKO.1-puro, Sigma-Aldrich), gag/pol-plasmid (psPAX2, addgene, Watertown, MMA, USA #12260) and VSV g envelope plasmid (pMD2.G, addgene #12259) into HEK293T cells. Viral supernatants were collected after 16 h and additionally after 24 h before pooling and mixing with a fresh medium (ratio: 1:1) supplied with 8 µg/mL protamine sulfate (Sigma-Aldrich) immediately prior to transduction. To select for positively transduced cells, 2 µg/mL and 0.5 µg/mL puromycin were added and maintained in the culture 48 h post-transduction for NCH644 and GS-5, respectively. pLKO.1-puro plasmids containing the shCHRDL1 or shCtrl (SHC002)-sequences were purchased from Sigma-Aldrich. To deplete CHRDL1, one of the following sequences was used:
3′-CCGGGAGAACTGTCATGGGAACATTCTCGAGAATGTTCCCATGACAGTTCTCTTTTTTG-5′(TRCN0000149739) and
3′-CCGGACGCCATGCACAGCATAATTTCTCGAGAAATTATGCTGTGCATGGCGTTTTTTG-5‘(TRCN0000371790) for NCH644 and GS-5, respectively.

sgRNAs were designed using Benchling (Uniyal et al, 2019 ; Biology Software) and cloned in the lentiCRISPR v2 plasmid (Addgene, #52961). Selected cells were isolated and cultured as single‐cells and screened by WB. Knockout clones identified by WB were further verified by Sanger sequencing. sgRNAs used are listed in Appendix Table S1.
shβCat (pLKO.1‐Hygro, Sigma, Mission shRNA ID #TRCN0000314921 (Aulicino et al, 2014 (link))), shKaiso (pLKO.1‐Puro, Sigma, MissionshRNA ID #TRCN000017838) or scrambled control (shControl; pLKO.1‐Puro, Sigma, Mission shRNA ID #SHC016) were used to transduce T‐ALL cells using lentiviral particles and selected with Hygromycin 800 μg μl⁻¹ (Invivogen) or with Puromycin 1.25 μg ml⁻¹ (Sigma).

Bacterial glycerol clones containing lentiviral plasmid vector pLKO.1-puro with shRNA specific for the α2-integrin subunit and pLKO.1-puro lentiviral vector with scramble shRNA were purchased from Sigma. Lentivirus was produced in HEK293T cells by co-transfection with shRNA-containing or scrambled shRNA-containing vectors with packing plasmids as described earlier [41 (link)]. Cells were transduced with lentivirus in the presence of polybrene (8 μg/ml) and selected with puromycin (1–2 μg/ml) for 2–4 days.