lcl pellets  (Qiagen)

Journal: medRxiv

Article Title: Duplication within 14q32.13 implicates a chimeric CLMN :: SYNE3 RNA transcript in cerebellar ataxia

doi: 10.64898/2026.04.23.26350376

Figure Lengend Snippet: (A) An IGV screen shot of LCL RNA-seq data shows an example of transcription (reads in blue) on the reverse strand of chromosome 14 in the intergenic space commencing from the 3’ breakpoint of the NC_000014.9:g.95239046_95570197dup variant downstream from GLRX5 three individuals from SCA30 that was not detected in any unrelated control. (B) Diagrammatic representation of SYNE3, CLMN and the CLMN::SYNE3 proteins overlaid with their respective exon structure. CLMN is a 1003 aa protein with two calponin homology domains (CH) indicated in blue. SYNE3 encodes a 976 aa protein with 8 spectrin-like repeats (SR) indicated with the darker pink circles and a C-terminal KASH domain (K) indicated in purple. The CLMN::SYNE3 protein is a fusion of exon 1 of CLMN to exon 2 of SYNE3 as shown. The amino and carboxyl termini are indicated with N and C respectively. (C) Differential gene expression analysis shows GLRX5 expression was upregulated in SCA30 samples (Log2 fold change 0.99, FDR=0.0039). CLMN, SYNE3, and DICER1, which closely neighbours the SCA30 duplication, showed no significant changes to their expression between SCA30 family members and unrelated controls. (D) Heat map showing expression of SYNE1, 2, and 3, and CLMN, across multiple developmental and post-natal time points in the human brain. The cerebellum shows an increase in CLMN expression with time, and a reduction in SYNE3 expression in adults. Weeks post-coitum (WPC). (E) Heatmap showing gene expression of Clmn, Syne1, 2, and 3, as average counts per nucleus per cluster, in mouse brain snRNAseq data. Syne1 has a general neuronal expression pattern, whereas Syne2 and Syne3 show a non-neuronal pattern. Clmn is expressed in thalamic projection neurons in the thalamus and Purkyně neurons in the cerebellum.

Article Snippet: Total RNA was extracted from LCL pellets using a QIAshredder (Qiagen: 79654) to homogenise, and RNeasy mini kit (Qiagen: 74106) to purify RNA, following manufacturer’s instructions.

Techniques: RNA Sequencing, Variant Assay, Control, Gene Expression, Expressing

Journal: medRxiv

Article Title: Duplication within 14q32.13 implicates a chimeric CLMN :: SYNE3 RNA transcript in cerebellar ataxia

doi: 10.64898/2026.04.23.26350376

Figure Lengend Snippet: PCR products for (A) CLMN :: SYNE3 using primers CLMN_Ex1_F and SYNE3_Ex4_R and (B) ESD derived from cDNA prepared from total RNA extracted from LCL of four female individuals who are not affected by SCA and unrelated to the SCA30 family compared to four affected individuals from the SCA30 family (V.56, V.61, VI.16 and V.82). PCR products were only amplified from samples where the reverse transcriptase was included in the reaction to produce cDNA (+RT) and not where it was excluded (-RT) indicating that these products are derived from mRNA transcripts spanning multiple exons. The CLMN :: SYNE3 product corresponds with a predicted 683 bp spanning CLMN Exon 1 to SYNE3 Exon 4 was only observed in affected individuals from the SCA30 family. The 453 bp amplicon from the ubiquitously expressed ESD gene indicates cDNA template is present in all +RT samples. Molecular weight markers are 1kb Plus ladder (Thermo Fisher Scientific cat. # 10787018).

Article Snippet: Total RNA was extracted from LCL pellets using a QIAshredder (Qiagen: 79654) to homogenise, and RNeasy mini kit (Qiagen: 74106) to purify RNA, following manufacturer’s instructions.

Techniques: Derivative Assay, Amplification, Reverse Transcription, Molecular Weight