GSM5903730: NPL-shRNA replicate 1; Homo sapiens; RNA-Seq
Total RNA from gene specific-shRNA and control-shRNA expressing THP1 cells were isolated at baseline and 48hr PMA treated condition using RNAeasy kit (Qiagene). RNA samples were reverse transcribed using Qiagen QuantiTect reverse transcription kit (Qiagen) based on manufacturer's protocol. To confirm shRNA mediated downregulation, expression of NPL was measured in cDNA by quantitative real-time PCR (qRT-PCR) using Power SYBR green chemistry (Applied Biosystems, Inc., Foster City, CA). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit II for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).