Minelute pcr purification column

ATAC-seq was performed as described previously (32 (link)) with minor modifications. Briefly, 5 × 10⁴ cells were pelleted at 300 g for 5 min, washed with 50 μl PBS and pelleted for 5 min at 300 g. Cells were lysed by resuspension in 50 μl of ATAC-seq RSB (10 mM Tris–HCl pH 7.4, 10 mM NaCl and 3 mM MgCl₂) containing 0.1% NP40, 0.1% Tween-20 and 0.01% digitonin and incubation on ice for 3 min. Nuclei were washed to remove contaminating mitochondria with 1 ml of RSB containing 0.1% Tween-20 and pelleted at 500 g for 10 min. Nuclei were then resuspended in 50 μl of transposition mix (25 μl 2× TD buffer, 2.5 μl transposase, 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20 and 5 μl water) and incubated on a thermomixer at 37°C for 30 min at 900 rpm. Reactions were purified using a Qiagen MinElute PCR-purification column. Library preparation was performed as described previously (33 ) with 10 cycles of amplification and purification using a Qiagen MinElute PCR-purification column. Samples were paired-end sequenced by Novogene on a NovaSeq 6000 S4 flow cell with a read length of 150 bp.

The ATAC-Seq experiments were performed as described previously^{37 (link)}. We performed two biological replicates per condition. Briefly, 50,000 cells were used per reaction. Nuclei were isolated after resuspension and centrifugation in Lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 1% (v/v) Igepal CA-630). The 50 µl transposase reaction with isolated nuclei (plus 25 µl TD, 2.5 µl TDE1 and 22.5 µl H₂O) was incubated at 37 °C for 30 min. DNA was purified using a MinElutePCR purification column (Qiagen). The transposed DNA fragments were preamplified by a first PCR reaction with five cycles containing barcoded Nextera PCR primers. The optimal number of cycles was determined by a SybrGreen qPCR reaction containing a 5-µl aliquot from the first PCR. The second PCR was then carried out with eight cycles and the libraries were first purified by MinElutePCR purification column (Qiagen) and then further size-selected by AMPure XP beads to obtain libraries with a size distribution between 150 and 1000 base pairs.

SABER-FISH probes targeting the genomes of AAV8-CMV-GFP, AAV8-Best1-sCX3CL1, and AAV8-CMV-H2B-FusionRed were designed in silico using ApE software. All probes were generated to recognize only the negative-sense strand of the vector. For each vector sequence, we first identified a list of non-overlapping oligonucleotides that were (1) complementary to the genome, (2) at least 36 nt long, and (3) predicted to have a melting temperature between 67°C and 71°C based on ApE’s default parameters. A 9-nt primer (5′-CATCATCAT-3′ or 5′-CAACTTAAC-3′) with a TTT linker was appended to the 3′ ends of these oligonucleotides to finalize the probe sequences, which were then ordered from Integrated DNA Technologies with standard desalting in a 96-well plate at the 10 nM synthesis scale. Probes were pooled into a single tube with a multi-channel pipette and extended to ∼500 nt using a previously described primer exchange reaction, in which a catalytic hairpin (Table S5) and strand-displacing polymerase repeatedly added the same sequence to the end of the 9-nt primer.¹³ (link) Extended probe sets were subsequently purified with MinElute PCR purification columns (QIAGEN) and eluted in nuclease-free water. Probes targeting Grik1 were designed as previously reported with sequences listed in Kishi et al.¹³ (link) Sequences for all other probes used in this study can be found in Tables S1–S4.

Isolated RNA (10 μg) was used for Random Primer cDNA synthesis using SuperScript II™ Reverse Transcriptase (Invitrogen Life Technologies 18064-071). The reaction mixture was treated with 1N NaOH to degrade any remaining RNA and treated with 1N HCl to neutralize the NaOH. Synthesized cDNA was then purified using MinElute® PCR Purification columns (Qiagen® 28004). Purified cDNA concentration and purity were measured using a Thermo Scientific NanoDrop™ 1000.