概要: Alternative splicing (AS) generates isoform diversity critical for cellular identity and homeostasis, yet characterization of this diversity in single cells remains limited. We developed Expedition, a computational framework to categorize and visualize the heterogeneity of AS from single-cell transcriptomes. Expedition consists of (i) outrigger, a de novo splice graph transversal algorithm to detect AS from single cell RNA-seq; (ii) anchor, a Bayesian approach to assign splicing modalities and (iii) bonvoyage, using non-negative matrix factorization to visualize modality changes. By applying Expedition to single iPSCs undergoing neuron differentiation, we discover that 25% of AS exons exhibit bimodality and are flanked by longer and more conserved introns harboring distinct cis-regulatory motifs. Bimodal exons are highly dynamic during cellular transitions, preserve translatability, enriched in recently emerged genes and have conserved AS in mammals. Applying Expedition (http://github.com/YeoLab/Expedition) in single cells redefines our estimates and understanding of AS in evolution and biology.
项目整体设计: Analysis of alternative splicing changes across motor neuron differentiation
iPSCs were cultured on matrigel coated plated using mTeSR(Stem Cell Technologies) media with mTeSR supplement at 37C incubator with 5% CO2.
处理方案:
Neuron progenitor cells were differentiated from iPSCs. Briefly, iPSCs were cultured in Matrigel coated plates and dislodged by dispase. To form embryonic bodies, the dislodged colonies were cultured in DMEM/F12(invitrogen) with GlutaMax and N2 supplement in non-adhere petri dish. Media were replaced every other day for 7 days. EBs were then placed onto matrigel coated plate to allow rosette formation. Clean rosette were picked manually and maintained in EB media for 7 days and subsequently dissociated with accutase and cultured in NPC media (DMEM/F12, GlutaMax, N2 and B27 with 2ug/ml FGF) to allow neuron progenitor cell differentiation. NPCs were maintained in NPC media.
提取方案:
Motor neurons were directly differentiated from iPSCs as previous described (Chambers S, Nature Biotechnology, 2009). Briefly, iPSCs were cultured on matrigel coated plates until fully confluent in mTeSR then switch to knock-out serum replacement media (KSR) containing Dorsomorphin(1uM) and SB431542(10uM). Upon day 4 of differentiation, increasing amounts of N2 media (25%, 50%) was added to the KSR. From day 7 of differentiation, 1.5uM retinoic acid and 200nM Smoothened Agonist (SAG, EMD Millipore) were added to induce patterning. Cells were dissociated on day 17 of differentiation and replated in poly-D-lysine and laminin coated plates. Maturation was performed using BDGF(2ng/ml), GDNF(2ng/ml), CNTF(2ng/ml), ascorbid acid, sonic hedgehog and retinoic acid in N2 and B27 media up until 35 days of differentiation.
建库方案:
Single cells were captured on C1 auto prep platform (Fluidigm, CA) according to manufacturer's instructions. C1 auto prep chips were visually inspected with a light microscopy at 20X to ensure singularity of captured cells. All non-single cells were discarded from analysis. SMARTer Ultra Low RNA cDNA Synthesis Kit (Clontech) was used to reverse transcribe polyA-tailed RNA. cDNA was amplified using Advantage 2 Polumerase Mix by PCR at 95¡C for 1 minutes, followed by 21 cylcles of 15 secondes at 95¡C, 30 seconds at 65¡C and 6 minutes at 68¡C, followed by another 10 minutes at 72¡C as a final extension. cDNAs were inspected using Agilent Bioanalyzer High Sensitivity DNA chips and quantitated by PicoGreen dsDNA Assay kit (ThermoFisher). cDNAs were diluted to 1ng to generate libraries using the Nextera XT DNA kit(Illumina, CA). Libraries were multiplexed and sequenced on Illumina HiSeq200 to generate 100bp PE reads.
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
paired; single
库的链类型:
-
测序平台:
Illumina
测序仪型号:
Illumina HiSeq 2000
链特异性:
Unspecific
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文章
Single-Cell Alternative Splicing Analysis with Expedition Reveals Splicing Dynamics during Neuron Differentiation.