wildtype wt thp1 cells  (ATCC)

Journal: bioRxiv

Article Title: CRISPRi Screen Identifies a Novel Growth Suppressor lncRNA, INSTAR , in Human Monocytes

doi: 10.1101/2025.10.30.685589

Figure Lengend Snippet: (A) Overview of CRISPRi THP-1 dropout screen. LncRNA transcription start sites (TSSs) were predicted using ChIP-Gro-Seq, RNA-sequencing, and FANTOM TSS data. NFkB-EGFP-CRISPRi-THP1 cells were subsequently infected with a pooled sgRNA library comprising over 2,000 sgRNAs targeting Gencode hg19 annotated lncRNA TSSs. After puromycin selection, samples were collected on day 21, and sgRNAs from the original plasmid library and day 21 were PCR amplified and sequenced. (B) CRISPRi THP-1 dropout screen analysis. Mann-Whitney U analysis was performed on each of the three screen replicates to determine sgRNA enrichment. Genes with MWU scores of -3 and 3 were considered significant. (C) Significant growth suppressor hits. The average log2 fold-change of the top three best-scoring sgRNAs for all significant hits with SD. Hits are color-coded to match their gene category in D. (D) Dropout screen hit categories. Significant hits were categorized based on gene/ lncRNA type using the UCSC Genome Browser (Human hg38). (E) Top 3 hits table. This table summarizes the top 3 lncRNA growth suppressor hits, INSTAR and STARD7-AS1 , and the positive control hit, PVT1 .

Article Snippet: Wildtype (WT) THP1 cells were obtained from ATCC.

Techniques: RNA Sequencing, Infection, Selection, Plasmid Preparation, Amplification, MANN-WHITNEY, Positive Control

Journal: bioRxiv

Article Title: CRISPRi Screen Identifies a Novel Growth Suppressor lncRNA, INSTAR , in Human Monocytes

doi: 10.1101/2025.10.30.685589

Figure Lengend Snippet: (A) sgRNAs targeting INSTAR . The browser track displays the TOPO-TA determined dominant isoform of INSTAR in THP-1 cells in blue and the 3 sgRNAs (sgRNA 1, sgRNA 2, and sgRNA 3) designed to knock down INSTAR . Browser tracks include: isoforms in primary human macrophages, RNA-sequencing (GSE150571), and ATAC-sequencing reads (GSE96800) of the INSTAR locus in wildtype THP-1 cells. (B) Overview of THP-1 cell proliferation assay. The top three sgRNAs were cloned into a plasmid with an mCherry fluorescent reporter, followed by lentivirus infection and puromycin selection of dCas9-KRAB THP1 cells. Mcherry-positive cells were mixed with mcherry-negative cells in a 1:1 ratio and plated in triplicate for each sgRNA for each gene. Cells were flowed every week for a total of 35 days. (C) CRISPRi knockdown of INSTAR in THP-1 cells. RT-qPCR of INSTAR across three biological replicates shows a statistically significant knockdown of INSTAR by all three sgRNAs vs. a non-targeting sgRNA (Neg Ctrl). Values are normalized to HPRT. (D) THP-1 cell competition assay results for INSTAR -KD. We monitored the proliferation of sgRNA-edited (mCherry-positive) cells compared to unedited (mCherry-negative) cells over 35 days, starting with a 1:1 mixture. The experiment was repeated 3 times, and a representative experiment is shown. Rate of change was calculated as the percentage difference in the averaged fold change between the knockdown cell lines and the non-targeting control.

Article Snippet: Wildtype (WT) THP1 cells were obtained from ATCC.

Techniques: Knockdown, RNA Sequencing, Sequencing, Proliferation Assay, Clone Assay, Plasmid Preparation, Infection, Selection, Quantitative RT-PCR, Competitive Binding Assay, Control