Kapa hifi hot start kit

Stage 0 iNKT cells (PBS57–CD1d tetramer⁺, CD44⁻, CD24⁺, CD69⁺, CD4^lo/−, CD8⁻) were sorted from the pooled thymi of three BALB/c or SKG mice (~1 × 10³ cells were recovered) directly into Trizol buffer. Three independents samples were generated for each genotype. RNA quality was evaluated with the Bioanalyzer RNA pico kit (Agilent Technologies). Libraries were prepared from polyA⁺ RNA using the Whole Transcriptome Amplification Sequencing Technology SEQR kit (Sigma-Aldrich). Briefly, purified RNA was reverse transcribed using SuperScript II, Oligo dT30 VN primers, and template switching primers. A preamplification step of eight PCR cycles was performed using the Kapa HiFi Hotstart kit (Kapa Biosystems). The PCR product was purified using AMPureXP beads (Beckman Coulter) and 1 ng was further used for library preparation using the Nextera XT LibraryPrep kit (Illumina). Tagmented DNA was amplified with a 12-cycle PCR and again purified with AMPureXP beads. Library size distribution and yield were evaluated using the Bioanalyzer high-sensitivity DNA kit. Libraries were pooled at equimolar ratios and sequenced with the rapid run protocol on a HiSeq. 2500 (Illumina) with 50-nt single end cycling.

RNA-seq was done using a Smart-seq2 protocol as described (Picelli et al., 2014 (link)). After neuron isolation by FACS, cDNA was prepared from each sample by reverse transcription using SuperScript II reverse transcriptase (18064-014, Invitrogen), Oligo-dT₃₀ and Template-Switching Oligonucleotide (TSO) primers listed in Table S2. After the first strand reaction, the cDNA was amplified with the KAPA Hifi HotStart kit (KK2601, KAPA Biosystems) and IS PCR primers listed in Table S2. cDNA was then purified using Ampure XP beads (A 63881, Beckman Coulter), tagmented and 1 μg was used for preparing libraries with the Illumina Nextera XT DNA sample preparation kit (FC-131-1096, Illumina), as per manufacturer suggested practices. Sequencing libraries were then submitted for sequencing on the Illumina HiSeq 4000 platform.

ATAC-seq was carried out as described earlier with minor modifications (27 ). Cells were scraped and counted to achieve 50 k/ml in ice-cold phosphate-buffered saline. Cell suspension was further diluted to 25 k/ml and nuclei were isolated with ATAC-LB (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL). Nuclei from 25 k cells from two biological replicates were used for tagmentation using Nextera DNA Library Preparation Kit (Illumina). After tagmentation, DNA was purified with Minelute PCR Purification Kit (Qiagen). Tagmented DNA was amplified with Kapa Hifi Hot Start Kit (Kapa Biosystems) using 16 polymerase chain reaction (PCR) cycles. Amplified libraries were purified again with Minelute PCR Purification Kit. The fragment size distribution of libraries was assessed with Agilent Bioanalyzer and sequenced on a HiSeq 2500 platform.

Mutations in exons 40–41 region (nt 13535–13756, GenBank # NC_000016.10) and exon 6 splicing site (nt 46232–46500, GenBank # NC_000016.10) of the TSC2 gene were screened in TSC2^NEG, TSC2^POS cell lines and in LAM-derived primary cells as previously reported^{29 (link)}. Genomic DNA was extracted with Quick DNA Universal Kit (Zymo Research) and amplified with the “KAPA HIFI Hotstart kit” (Kapa Biosystem). PCR products were purified with the “Zymoclean Gel DNA recovery” kit (Zymo Research), sequenced (GATC, France) and analyzed with the Vector NTI software (Invitrogen).