For all the oligonucleotide primers used during cloning, please refer to data S1J. Note that all plasmids were confirmed by sequencing and, upon reasonable request, they are available from the authors. The bidirectional pBI-CMV1 vector was purchased from Clontech (CA, USA) and modified with the QuikChange II Site-directed mutagenesis kit (Agilent, CA, USA) to delete the Bgl II restriction site within the multiple cloning site no. 1 (MCS1). The VAMP-2 pHluorin sequence was designed in silico on the basis of a previous publication (15), and it was ordered at GenScript (USA) and inserted in the MCS1 upon digestion with Kpn I and Not I. The plasmid encoding for the tetanus toxin light chain (pGEMTEZ-TetTxLC) was a gift from R. Jahn. TetTxLC or the six lncRNAs (data S1B) were inserted in the MCS2 of the pBI-CMV1-pHluorin plasmid with a PCR strategy upon either pGEMTEZ-TetTxLC or Rat genomic DNA amplification. The cloning primers used in the PCR strategy for insertion into the MCS2 added the following couples of restriction sites during amplification: Bcl I/Spe I (for TetTxLC and uc003wst.1), Bgl II/Xba I (uc021rvu.1, uc004bbl.1 and uc003jrq.3), Bgl II/Spe I (uc001pyz.3), or Bam HI/Xba I (uc004bdv.3). The X1 variant of rat TDP-43 (NCBI Reference Sequence: XM_006239382.3) was PCR-amplified from rat complementary DNA (cDNA) to add the Xba I and Sac II restriction sites and was inserted into the pBI-CMV1-pHluorin plasmid construct. For cloning of TDP-43-AS, the PCR fragment was inserted in antisense orientation using Bsu36 I and Sac II restrictions. The plasmid ensured an efficient reduction in TDP-43 expression levels with respect to control-transfected C6 cells as tested by qRT-PCR (fig. S5E). For in utero overexpression, neuroLNC was subcloned into the pCAGEN vector (obtained from Addgene) with a PCR strategy adding the Xho I and the Not I restriction sites. For viral overexpression, neuroLNC was cloned into an AAV backbone that was previously described (40) allowing the specific expression in neurons (through the human synapsin-1 promoter). For cloning in the viral vector, we used a PCR strategy allowing the addition of the restriction sites Asc I (also known as Sgs I) and Sbf I (also known as Sda I). Positive viral clones were always tested for the integrity of their inverted terminal repeats. For down-regulation, shRNAs against rat and mouse sequences were designed using the Biosettia tool (available at http://biosettia.com/support/shrna-designer/, Biosettia, CA, USA) and cloned following the manufacturer’s instructions into the pRNA1-M6-green plasmid [Biosettia; expressing green fluorescent protein (GFP) concomitantly with the shRNA]. As a negative control, the scramble vector provided by Biosettia was used. Note that among several tested down-regulation methods only the use of the mouse U6 pol-III promoter for the expression of shRNAs was efficiently down-regulating neuroLNC. The neuroLNCMUT sequence was designed in silico (as specified in data S1I) and was ordered at GenScript (USA). For all subclonings, we relied on commercial chemically competent bacteria (α-select bronze competent cells, Bioline or SURE bacteria from Agilent in case of adeno-associated viral constructs). For transformation, 50 μl of bacteria was heat-shocked at 42°C for 35 s and cooled briefly before addition of 400 μl of Super Optimal broth with Catabolite repression (SOC) medium (Sigma-Aldrich). After incubation for 45 min at 37°C and shaking at 300 rpm, cells were centrifuged and plated on agar plates with the appropriate antibiotic selection. Colonies were picked on the following morning and grown in LB. Midi preps were prepared using the Macherey-Nagel NucleoBond Xtra Midi EF Kit according to the low copy number protocol. DNA was eluted in double-distilled ultrapure water (ddH2O). Quality and amount of DNA were assessed by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).

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