Vsv g

For generation of DC2.4 or THP-1 cells with knockdown of LMAN1, shRNA against mouse or human LMAN1 in the pLKO.1 lentiviral system was used (Horizon Discovery; Boyertown, PA) (Cat# RMM3981-201783827 (mouse) and Cat# RHS3979-201775306 (human)). For generation of DC2.4 and THP-1 cells with overexpression of LMAN1, pCMV3 SP-N-HA-mouse LMAN1(Sino Biologicals; Wayne, PA) (Cat# MG5A0204-NY) or pCMV3 human-SP-N-HA-human LMAN1 (Sino Biologicals; Wayne, PA) (Cat# HG161166-NY) were subcloned into the pBABE puro vector (Addgene; Watertown, MA) (Cat#1764). For retroviral transduction, pBABE plasmids were used with pUMVC (Addgene; Watertown, MA) (Cat# 8449) and VSV-G (Addgene; Watertown, MA) (Cat# 8454) to generate viral particles. For lentiviral transduction, pLKO plasmids were used with psPAX2 (Addgene; Watertown, MA) (Cat# 12260) and pMD2.G (Addgene; Watertown, MA) (Cat# 12259) to generate viral particles. Viral transduction was carried out for 48hrs and cell selection was carried out using Puromycin.

SMPD3 CRISPR/Cas-9 knockout cells were generated using the guidelines provided by www.crispr.mit.edu. Guide oligos were designed using the MIT CRISPR database. The oligo and complement chosen were SMPD3-F 5′-CACCGTGGCGCTTCTCGTAGGTGGT-3′ and SMPD3-R 5′- AAACACCACCTACGAGAAGCGCCAC-3′. The oligos were cloned into LentiCRISPR.v2 (AddGene). Lentivirus was produced using LentiX 293T cells and the packaging plasmid pCMV-dr8.91 (AddGene) and envelope plasmid VSV-G (AddGene) with the designed guide DNA LentiCRISPR plasmid. Lentivirus was filtered through 0.45μm filter (Millipore) and supplemented with 8 μg/mL polybrene. Complete lentivirus was used to transduce SVG-A with three serial 24 hour infections. Transduced cells were then selected with 2 μg/mL puromycin. This is the mixed population of CRISPR clones. Single clones were picked using cell rafts (Cell Microsystems) and the QIAscout system (QIAGEN). Clones were expanded from a 96 well plate and sequences confirmed according to MGH CRISPR Amplicon sequencing guidelines (www.dnacore.mgh.harvard.edu). Primer pair sets for the SMPD3 gene are as follows: Fwd1 5′-GTGTGTCCTGGGCCCTTATC-3′, Rev1 5′-GGGGACCAGAAGAGAAAGCC-3′ and Fwd2 5′-CTAACAGCTGTCTGTCCGCC-3′, Rev2 5′-AGAGAAAGCCGAGAAACGCA-3′.