Hiseq 3000 4000 sbs kit

RNA from flow-sorted CD31/CD45⁻ cells from freshly isolated, 5 male C57Bl/6 mice-derived leptomeningeal tissue was extracted using RNeasy Micro Kit (Qiagen, #74034). After RiboGreen quantification and quality control by Agilent BioAnalyzer, 2 ng total RNA with RNA integrity numbers ranging from 9.7 to 10 underwent amplification using the SMART-Seq v4 Ultra Low Input RNA Kit (Clonetech catalog # 63488), with 12 cycles of amplification. Subsequently, 10ng of amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) using 8 cycles of PCR. Samples were barcoded and run on a HiSeq 4000 in a 50bp/50bp paired end run, using the HiSeq 3000/4000 SBS Kit (Illumina). An average of 36 million paired reads were generated per sample. Reads from generated FASTQ files were quality checked and mapped to the mouse reference genome (mm10) using STAR2.5.0.a. The expression count matrix of uniquely mapped reads was computed with HTseq v0.5.3. Raw counts were normalized by library size using DESeq2 pipeline in R v3.6.0 running in RStudio v1.0.143 (see Table S1). The single-cell data from the Allen Brain Map were analyzed via UCSC Cell Browser (mouse cortex, v2019/2020, 75k cells, accessed in May 2020; see Tables S2–3) ^{32 (link),33 (link)}. Transcriptomic signatures of VLMC clusters were determined with Reactome ^{34 (link)}.

RNA sequencing libraries were constructed from total RNA using the KAPA Stranded mRNA kit and sequenced on an Illumina Hi Seq 3000 using the Hi Seq 3000/4000 SBS kit (50 cycles). Raw reads were aligned to the UCSC Human Genome hg19 using the Bowtie2 aligner (version 2.2.9) (RRID:SCR_016368). Read count was performed by RSEM (version 1.2.25) (RRID:SCR_013027). Genes with less than 10 fragments in all samples were excluded from differential expression analysis determined using the DEseq2 pipeline (version 2.6.10) (RRID:SCR_015687). FDR cutoff of < 0.05 was considered significant. Gene set enrichment analysis (GSEA, v2.2.3) (RRID:SCR_003199) was performed on ranked lists of differentially expressed genes using the GSEAPreranked tool and Hallmark, C2:CP:KEGG gene set categories obtained from the Molecular Signatures Database. Default GSEA parameters were used with the exception of the classic enrichment statistic.

RNA was isolated from sorted cell populations with TRIzol (Invitrogen), which was followed by SMARTer amplification. Paired-end Illumina next-generation sequencing was performed for RNAseq (70 (link)). DNA was prepared from sorted cells for ATACseq. Libraries were sequenced on a HiSeq 2500 in High Output mode and a HiSeq4000 in a 50bp/50bp paired end run, using the TruSeq SBS Kit v4 or HiSeq 3000/4000 SBS Kit (Illumina) as previously described (70 (link)).

ATAC-seq libraries were prepared as described (Buenrostro et al., 2015 ). 5x10⁴ cells were washed in ice cold PBS and lysed using ATAC-seq lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Following lysis, cells were incubated in 1x transposition buffer (Nextera TD buffer) containing 2.5 μL Nextera Tn5 transposase for 30 minutes at 37 degrees. Transposed DNA fragments were isolated using Qiagen MinElute Reaction Cleanup kit and amplified using barcoded primers with Illumina adapter sequences. The number of cycles used for library amplification was determined using a quantitative PCR side-reaction. Amplified libraries were purified and size selected using Ampure XP beads using consecutive purifications with bead-to-sample ratios of 0.5 and 1.8. After PicoGreen quantification and quality control by Agilent BioAnalyzer, libraries were run on a HiSeq 4000 in a 100bp/100bp paired end run, using the HiSeq 3000/4000 SBS Kit (Illumina). The average number of read pairs per sample was 31 million.