Gdna wipeout buffer

For isolation of total RNA, we used the RNeasy® Mini kit (Qiagen, Hilden, Germany) following the manufactures guide. 500 ng of total RNA were reverse transcribed using the Primescript® Reverse Transcription Kit (Takara, Tokyo, Japan) or the Quantitect Reverse Transcription Kit along with gDNA wipeout buffer (Qiagen).
AR-FL expression was analyzed using TaqMan PCR assay (Hs00171172_m1) (Thermo Fisher Scientific). AR-V status was analyzed by using previously described custom-made TaqMan PCR assays specific for detection of AR-Vs AR-V3, AR-V7 and AR-V9 [17 (link)]. Assay sequences are listed in Table S1.
All qPCR runs were performed along with TaqMan PCR assays for housekeeping genes RPL37A (Hs01102345_m1) and HPRT1 (Hs99999909_m1) (Thermo Fisher Scientific). qPCR reactions were run using the Luna Mastermix on a QuantStudio 3 qPCR cycler (Thermo Fisher Scientific).

Total RNA (900 ng) purified from A. baumannii was incubated for 5 min at 42°C with gDNA Wipeout Buffer (Qiagen). The resulting RNA was converted into cDNA using the QuantiTect Reverse Transcription kit with random primers and Quantiscript Reverse Transcriptase. Samples were incubated for 30 min at 42°C as per the manufacturer’s instructions (Qiagen). cDNA reactions were then diluted 1:10 with sterile water. Oligonucleotide primer pairs for quantitative real-time PCR (qRT-PCR) were generated using the Primer-BLAST server.³ Primers were designed to amplify ∼100–200 bp fragments for each gene. qRT-PCR was performed using PowerSYBR Green PCR Master Mix (Thermo Fisher Scientific) with a Biorad CFX-96 cycler. The following cycling parameters were used to amplify and quantify the fragments: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 60 s. Melting-curve data were collected to ensure proper amplification of target genes. Data were generated from three separate RNA isolations, cDNA preparations, and from technical triplicates for each primer set. The relative expression of each gene was determined by comparing target gene expression with reference gene clpX as previously described (Tipton and Rather, 2017 (link)).

Cells were lysed with 350 μL RLT Buffer (QIAGEN GmbH, Germany) per well with 1% β-mercaptoethanol (Sigma-Aldrich, Germany). RNA was automatically isolated from cell lysates with the QIACube (QIAGEN GmbH, Germany) using QIAgen miRNeasy Kit (QIAGEN GmbH, Germany). Isolated RNA was measured with a NanoDrop^TM 3300 Fluorospectrometer (Thermo Fisher Scientific, United States) and diluted, respectively, with RNAse free water (QIAGEN GmbH, Germany) to a final concentration of 41.67 ng/μL. Further 2 μL gDNA Wipeout Buffer (QIAGEN GmbH, Germany) was added to 12 μL sample, equaling 500 ng RNA, and incubated at 42°C for two minutes. Reverse Transcriptase MM, QuantiScript RT Buffer and RT Primer Mix from the QuantiTect Reverse Transcription Kit (QIAGEN GmbH, Germany) were then added to the samples, which were then incubated at 42°C for 15 min for cDNA synthesis. The Reverse Transcriptase was then inactivated via incubation at 95°C for 3 min.

A total of 1000 sorted pure CD4⁺TIM-3⁺, CD4⁺TIM-3⁻, and CD4⁺CD25⁺ T cells from TT were used to generate cDNA libraries for RNA-Sequencing using QIAseq FX Single Cell RNA Library Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Briefly, sorted cells were spun down and lysed immediately. The gDNA was then removed by using gDNA Wipeout buffer (Qiagen). The gDNA-removed lysates were used to generate double-stranded DNA, which were subsequently amplified using REPLI-g sc SensiPhi DNA polymerase. Small fractions of amplified products were cleaned using PureLink PCR Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) and quality checked using Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA). The quality passed DNA (>2000 bp) were quantified using Qubit dsDNA HS assay kit (Invitrogen, Carlsbad, CA, USA). A total of 500 ng to 1 µg DNA was enzymatically fragmented and ligated using paired adaptors. DNA was further purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). The yield and size distribution of libraries (500–1000 bp) were determined using Qubit dsDNA HS assay kit (Invitrogen) and Agilent High Sensitivity DNA Kit (Invitrogen).

Total RNA samples were extracted from treated and control PIE cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with gDNA Wipeout Buffer (Qiagen, Tokyo, Japan). RNA was reverse transcribed using a Quantitect reverse transcription (RT) kit (Qiagen, Tokyo, Japan) according to the manufacturer’s recommendations. The synthesized cDNA was then used for quantitative PCR analysis using Platinum SYBR green qPCR SuperMix UDG with ROX (Invitrogen, Carlsbad, CA, USA) on 7300 real-time PCR system (Applied Biosystems, Warrigton, United Kingdom). The primers used for the analysis of IL-6, IFN-β, and β-actin were described in previous publications [21 (link),22 (link)]. PCR cycling conditions were 5 min at 50 °C, followed by 5 min at 95 °C, and then 40 cycles of 15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. Expression of β-actin in each sample was assessed, and the β-actin data were used as an internal control to normalize differences between samples and calculate relative expression levels. Samples were run in triplicate for each experimental condition, and mean values were used to calculate statistics.

For all qPCR experiments, mRNA was sourced from male Tc1 mouse hippocampus (n = 4, 6 months). For each sample (labeled WT/Tc1 A–D), 1 μg mRNA was first cleared of genomic DNA (gDNA) via incubation in 2 μl gDNA Wipeout Buffer (2.5–10% trometamol - QIAGEN) and 12 μl RNAse-free ddH₂0 at 42 °C for 2 min. Following, mRNA was reverse-transcribed into cDNA using a QuantiTect. Reverse Transcription Kit (QIAGEN) according to manufacturer’s instructions. The resulting ~1 μg (50 ng/μl) cDNA product was stored at −20 °C for experimental use. For each sample, an identical reaction was run alongside, substituting the reverse transcriptase with RNAse-free ddH20 for usage as a negative control (−RT) to account for any non-specific amplification at the qPCR stage.