GSM9267099: GFP-exon 6 XISTrep3; Homo sapiens; RNA-Seq
For ectopic GFP and GFP-short hXIST expression, HEK293T cells were transfected with 10mg of pLV-eGFP and pLV-eGFP-short hXIST lentiviral construct, 10mg packing plasmids (psPAX2, pMD2.G, from Addgene plasmids #12259 and #12260), and 40ml jetPEI transfection regent (Polyplus) according to the manufacturer's instructions. Cells were replaced with fresh DMEM medium after 24 hours post-transfection. Lentivirus-containing DMEM was collected 48 hours post-transfection and filtered through a 0.45mm filter. HEK293T was cultured with lentivirus-containing media and supplemented with 4mg/ml polybrene (Millipore Sigma). Virus-containing media were replaced 24 hours post-infection with fresh DMEM, and cells were cultured for an additional 24 hours. Virus-transfected cells were selected with 5μg/mL puromycin (Millipore Sigma) for 3 days. GFP and GFP-short hXIST-expressed cells were collected directly into RLT Plus buffer (QIAGEN), and total RNA was isolated with the RNeasy Plus Mini Kit (QIAGEN) and RNase-free DNaseI treatment (QIAGEN) according to the manufacturer's instructions. For bulk mRNA-seq library preparation, the total RNA quality was measured using a Bioanalyzer (Agilent), and samples with RNA integrity numbers (RIN) > 8 were used. Libraries were constructed from 100ng of total RNA using the Universal Plus mRNA-Seq with NuQuant® (Tecan). Following the manufacturer's instructions of mRNA elution, mRNA was subjected to fragmentation at 94°C for 8 minutes. First strand, second-strand cDNA synthesis, end repair, adapter ligation, and amplification were carried out by following the manufacturer's instructions. The concentration and quality of the libraries were determined using Qubit (Invitrogen) and Bioanalyzer (Agilent). Constructed mRNA-seq libraries were sequenced at GENEWIZ (Azenta) on the Illumina Nova-seq platform, obtaining 150-nucleotide paired-end reads.