kapa low throughput library preparation kit standard  (Roche)

Journal: mBio

Article Title: WDR81 represses IKK-mediated expression of pro-survival genes to regulate apoptosis

doi: 10.1128/mbio.02722-25

Figure Lengend Snippet: In the absence of WDR81, pro-survival gene expression is upregulated. ( A ) Total RNA was harvested from MEFs, and gene expression was analyzed by RNA-seq. A volcano plot of genes differentially expressed in WT and ΔWDR81 is shown. Genes that are differentially expressed with FDR <0.0001 are shown in red. ( B ) Genes in the volcano plot shown in A that are also differentially expressed in WT and TNF-treated WT cells are marked in blue. ( C ) Heat map of 66 genes that are differentially expressed both in ΔWDR81 and WT MEFs as well as in TNF-treated and control WT cells is shown. A scale bar showing log2 fold change in gene expression is included. ( D ) MEFs were grown in a 24-well plate, and total mRNA was extracted using Bio-Rad Aurum Total RNA mini kit according to manufacturer’s protocol. cDNA was synthesized using Applied Biosystems High-Capacity cDNA Reverse Transcription kit. qPCR for A20 gene expression was done using Applied Biosystems StepOnePlus Real-Time PCR system. A20 gene expression was calculated using the 2 -ΔΔCT method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. Error bars represent SD. ***, P < 0.0005 by Student’s t -test. ( E ) MEFs were grown in a 24-well plate, and total mRNA was extracted using Bio-Rad Aurum Total RNA mini kit according to manufacturer’s protocol. cDNA was synthesized using Applied Biosystems High-Capacity cDNA Reverse Transcription kit. qPCR for Bcl2 gene expression was done using Applied Biosystems StepOnePlus Real-Time PCR system. Bcl2 gene expression was calculated using the 2 -ΔΔCT method using GAPDH as internal control. Error bars represent SD. *, P < 0.05 by Student's t -test. ( F ) MEFs were grown in standard media as described in Materials and Methods. Total mRNA was extracted using Bio-Rad Aurum Total RNA mini kit according to manufacturer’s protocol. cDNA was synthesized using Applied Biosystems High-Capacity cDNA Reverse Transcription kit. qPCR for A20 gene expression was done using Applied Biosystems StepOnePlus Real-Time PCR system. A20 gene expression was calculated using the 2 -ΔΔCT method using GAPDH as internal control. Error bars represent SD. ***, P < 0.0005, **, P < 0.005, by one-way analysis of variance.

Article Snippet: Briefly, cDNA library construction was done using a TruSeq Stranded mRNA Low-Throughput Sample Prep Kit (Illumina) following the manufacturer’s protocol.

Techniques: Gene Expression, RNA Sequencing, Control, Synthesized, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: mBio

Article Title: WDR81 represses IKK-mediated expression of pro-survival genes to regulate apoptosis

doi: 10.1128/mbio.02722-25

Figure Lengend Snippet: Higher A20 mRNA levels in WDR81-deficient cells are driven by IKK but not PI3K signaling. ( A ) MEFs were incubated in media with either DMSO control, TNF (50 ng/mL), or IKK inhibitor (5 µM) for 3 h. Total mRNA was extracted using Bio-Rad Aurum Total RNA mini kit according to manufacturer’s protocol. cDNA was synthesized using Applied Biosystems High-Capacity cDNA Reverse Transcription kit. qPCR for A20 gene expression was done using Applied Biosystems StepOnePlus Real-Time PCR system. A20 gene expression was calculated using the 2 -ΔΔCT method using GAPDH as internal control. Error bars represent SD. ***, P < 0.0005 by one-way analysis of variance (ANOVA). ( B ) MEFs were incubated in media with DMSO control or PI3K inhibitor (25 µM) for 3 h. Total mRNA was extracted using Bio-Rad Aurum Total RNA mini kit according to manufacturer’s protocol. cDNA was synthesized using Applied Biosystems High-Capacity cDNA Reverse Transcription kit. qPCR for A20 gene expression was done using Applied Biosystems StepOnePlus Real-Time PCR system. A20 gene expression was calculated using the 2 -ΔΔCT method using GAPDH as internal control. Error bars represent SD. **, P < 0.005, *, P < 0.05 by one-way ANOVA.

Article Snippet: Briefly, cDNA library construction was done using a TruSeq Stranded mRNA Low-Throughput Sample Prep Kit (Illumina) following the manufacturer’s protocol.

Techniques: Incubation, Control, Synthesized, Reverse Transcription, Gene Expression, Real-time Polymerase Chain Reaction