TITLE: Cell line construction and maintenance for Lyso-IP with or without genes linked with lysosomal storage disease
AUTHORS: Sharan Sharan Swarup, Harper    JW

[DESCRIPTION]
Lyso-IP is a method that allows for the isolation of lysosomes for proteomics and metabolomics using HA-tagged TMEM192 (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.bx9hpr36).  Here, we describe methods for cell line construction and maintenance of HeLa cells with TMEM192-3xHA with or without deletion of genes linked with lysosomal storage diseases. 

[STEPS]
SECTION: Cell line maintenance
1. Maintain HeLa cells in Dulbecco’ Modifies Eagles Medium (DMEM) with 10% fetal bovine serum and optional 1% penicillin-streptomycin. 
SECTION: Endogenous tagging of TMEM192 with 3xHA
2. For endogenous tagging of TMEM192 with 3xHA, co-transfect HeLa cells with pX459 containing a gRNA (5’-AGTAGAACGTGAGAGGCTCA) targeting adjacent to the translational termination sequence in TMEM192 and pSMART containing 5’ and 3’ homology arms for TMEM192 in which the termination codon is replaced by a 3xHA epitope sequence followed by a TAA stop codon (Addgene #175777).
SECTION: Endogenous tagging of TMEM192 with 3xHA
3. Identify homozygously targeted clones by immunoblotting cell extracts with α-HA and α-TMEM192. These are referred to as HeLa-TMEM192-HA cells for Lyso-IP.
SECTION: Targeted knock-out specific genes including GRN, HEXA, NPC1 and NPC2
4. For GRN knock-out, phosphorylate and anneal oligonucleotides (Top: 5’- ATCGACCATAACACAGCACG, Bottom: 5’-CGTGCTGTGTTATGGTCGAT), and clone into a pX459 vector.  For HEXA knock-out, phosphorylate and anneal oligonucleotides (Top: 5’- CGGCCGAGCTGACATCGTAC, Bottom: 5’-GTAGCATGTCAGCTCGGCCG), and clone into a pX459 vector. For NPC1 knock-out, phosphorylate and anneal oligonucleotides (Top: 5’- TACCTGGACAGAAACTGTAG, Bottom: 5’-CTACAGTTTCTGTCCAGGTA), and clone into a pX459 vector. For NPC2 knock-out, phosphorylate and anneal oligonucleotides (Top: 5’- AGCTGCCAGGAAACGCATCG, Bottom: 5’-CGATGCGTTTCCTGGCAGCT), and clone into a pX459 vector. 
SECTION: Targeted knock-out specific genes including GRN, HEXA, NPC1 and NPC2
5. Transfect HELA-TMEM192-HA cells with the pX459-gRNA-APP plasmid (Addgene #176487) with Lipofectamine 3000, and select with 1.2 µg/mL of puromycin. Select monoclonal cells, and confirm target gene deletion by Western blotting, and/or Sanger sequencing of the edited alleles.
SECTION: Rescue of GRN expression
6. The entry vector pDONR223 containing full-length GRN open reading frame (1179 base pairs) is recombined with a pHAGE lentivirus destination vector using Gateway cloning technology (Thermo Fisher).
SECTION: Rescue of GRN expression
7. Make lentivirus for transduction of pHAGE-GRN by transfecing 293T cells along with psPAX2, pMD2.G (Addgene Cat#12260 Cat#12259) and pHAGE-GRN in a 4:2:1 ratio using polyethyleneimine. Virus-containing supernatant was harvested 2 days after transfection and filtered through a 0.45-micron syringe filter. Polybrene was added to a final concentration of 8 mg/ml to the viral supernatant. 
SECTION: Rescue of GRN expression
8. HeLa Tmem192-3xHA GRN KO cells were infected with 50 mL of viral supernatant, and stable cell lines were selected 48 h post-infection using hygromycin at a concentration of 100 mg/mL. 
SECTION: Rescue of GRN expression
8.1. Maintain HeLa cells in Dulbecco’ Modifies Eagles Medium (DMEM) with 10% fetal bovine serum and optional 1% penicillin-streptomycin with hygromycin at a concentration of 100 mg/mL.