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Pineal; Lhx4, Bsx (Rat)

Specimens

Animals and sample preparation: Sprague‐Dawley rats (adult male 150-250 g) were obtained from Charles River (Sulzfeld, Germany) or Janvier (Saint Berthevin Cedex, France). Animals were housed under a 12 L:12 D light:dark (LD) schedule and sacrificed by CO₂ anesthesia followed by decapitation. All animal experiments were in accordance with the guidelines of EU directive 2010/63/EU and approved by the Danish Council for Animal Experiments (authorization number 2017‐15‐0201‐01190) and the Faculty of Health and Medical Sciences, University of Copenhagen (authorization number P17‐311).

For each preparation of primary pinealocyte cell culures, 12 to 22 pineal glands were collected at ZT 4. Cultures were prepared as previously described with 105 cells per sample. For knockdown experiments, pinealocytes were transfected with one of three siRNAs (40 nmol/L) using Lipofectamine RNAiMAX (Life Technologies): 1) Lhx4, Stealth siRNA targeting position 1221‐1245 on NM_001108348.1 (siLhx4) (Life Technologies; #RSS323070); 2) Bsx, Stealth siRNA targeting position 192-216 on NM_001191995.1 (siBsx) (#RSS366168); or 3) nontargeting siRNA (siNT) (#12935300) was used as negative control. The medium was changed 24 hours after initiation of transfection. On the next day, pinealocytes were treated with DBcAMP (500 µmol/L) for 6 hr prior to being harvested. There are nine samples: triplicates of Control nontargeting siRNA (siNT), siRNA targeting Lhx4 (siLhx4), or siRNA targeting Bsx (siBsx).

RNA Preparation

RNA extraction: RNA was extracted using TriZOL (Life Technologies) and purified with a Qiagen RNeasy® Micro Kit including DNase I treatment (Qiagen). RNA quality was determined on a Bioanalyzer 2100 (Agilent) with RIN‐values ranging from 7.7 to 8.9.

RNA-Seq library preparation and sequencing

Library preparation and sequencing: The sequencing libraries were prepared using 3.4 ng total RNA with ribosomal RNA depletion employing a SMARTer® Stranded Total RNA‐Seq Pico Input Mammalian Kit (Takara Bio). Nine barcoded libraries were pooled and sequenced on one Flow Cell of a HiSeq2500 (Illumina) in Rapid Run Mode yielding a total of 410 million 2x100‐bp read‐pairs (average reads per sample: 45.6M ± 3.3M SEM). The data are available in NCBI GEO as GSE142476.

Bioinformatics methods

See bioinformatics methods for details.

References

  1. Hertz H, Carstensen MB, Bering T, Rohde K, Møller M, Granau AM, Coon SL, Klein DC, Rath MF. The Lhx4 homeobox transcript in the rat pineal gland: Adrenergic regulation and impact on transcripts encoding melatonin-synthesizing enzymes. J Pineal Res 2020 Jan;68(1):e12616. PMID: 31609018
  2. Carstensen MB, Hertz H, Bering T, Møller M, Rhode K, Klein DC, Coon SL, Rath MF. Circadian regulation and molecular role of the Bsx homeobox gene in the adult pineal gland. J Pineal Res 2020 Mar;68(2):e12629. PMID: 31808568

Samples

The following table describes the sample metadata for this experiment. Accessions in GEO, BioProject, and SRA link to their resepective entries. Raw data were downloaded as FASTQ format using fastq-dump for each of the SRR run accessions.

Note: scroll to the right to see the full extent of the table.

samplename BioSample Experiment SRRrun SRA_Sample Sample_Name SRA_Study BioProject LoadDate MBases MBytes Assay_Type Center_Name Consent DATASTORE_filetype DATASTORE_provider Instrument LibraryLayout LibrarySelection LibrarySource Organism Platform ReleaseDate AvgSpotLen source_name Age treatment_condition Strain tissue group
siNT-1 SAMN13663591 SRX7427147 SRR10752345 SRS5873232 GSM4230119 SRP238459 PRJNA597111 NaN 8717 3906 RNA-Seq GEO public fastq,sra gs,ncbi,s3 Illumina HiSeq 2500 PAIRED cDNA TRANSCRIPTOMIC Rattus norvegicus ILLUMINA 2019-12-24T00:00:00Z 197 adult pinealocytes adult siNT Sprague-Dawley pineal gland siNT
siNT-2 SAMN13663590 SRX7427148 SRR10752346 SRS5873236 GSM4230120 SRP238459 PRJNA597111 NaN 10514 4710 RNA-Seq GEO public fastq,sra s3,gs,ncbi Illumina HiSeq 2500 PAIRED cDNA TRANSCRIPTOMIC Rattus norvegicus ILLUMINA 2019-12-24T00:00:00Z 198 adult pinealocytes adult siNT Sprague-Dawley pineal gland siNT
siNT-3 SAMN13663589 SRX7427149 SRR10752347 SRS5873237 GSM4230121 SRP238459 PRJNA597111 NaN 10580 4686 RNA-Seq GEO public fastq,sra gs,s3,ncbi Illumina HiSeq 2500 PAIRED cDNA TRANSCRIPTOMIC Rattus norvegicus ILLUMINA 2019-12-24T00:00:00Z 198 adult pinealocytes adult siNT Sprague-Dawley pineal gland siNT
siLhx4-1 SAMN13663588 SRX7427150 SRR10752348 SRS5873233 GSM4230122 SRP238459 PRJNA597111 NaN 12279 5659 RNA-Seq GEO public sra,fastq s3,ncbi,gs Illumina HiSeq 2500 PAIRED cDNA TRANSCRIPTOMIC Rattus norvegicus ILLUMINA 2019-12-24T00:00:00Z 198 adult pinealocytes adult siLhx4 Sprague-Dawley pineal gland siLhx4
siLhx4-2 SAMN13663587 SRX7427151 SRR10752349 SRS5873234 GSM4230123 SRP238459 PRJNA597111 NaN 9147 4092 RNA-Seq GEO public fastq,sra ncbi,s3,gs Illumina HiSeq 2500 PAIRED cDNA TRANSCRIPTOMIC Rattus norvegicus ILLUMINA 2019-12-24T00:00:00Z 198 adult pinealocytes adult siLhx4 Sprague-Dawley pineal gland siLhx4
siLhx4-3 SAMN13663586 SRX7427152 SRR10752350 SRS5873235 GSM4230124 SRP238459 PRJNA597111 NaN 6328 2844 RNA-Seq GEO public sra,fastq s3,ncbi,gs Illumina HiSeq 2500 PAIRED cDNA TRANSCRIPTOMIC Rattus norvegicus ILLUMINA 2019-12-24T00:00:00Z 198 adult pinealocytes adult siLhx4 Sprague-Dawley pineal gland siLhx4
siBsx-1 SAMN13663585 SRX7427153 SRR10752351 SRS5873238 GSM4230125 SRP238459 PRJNA597111 NaN 6979 3274 RNA-Seq GEO public sra,fastq s3,ncbi,gs Illumina HiSeq 2500 PAIRED cDNA TRANSCRIPTOMIC Rattus norvegicus ILLUMINA 2019-12-24T00:00:00Z 198 adult pinealocytes adult siBsx Sprague-Dawley pineal gland siBsx
siBsx-2 SAMN13663584 SRX7427154 SRR10752352 SRS5873239 GSM4230126 SRP238459 PRJNA597111 NaN 9101 4097 RNA-Seq GEO public fastq,sra gs,s3,ncbi Illumina HiSeq 2500 PAIRED cDNA TRANSCRIPTOMIC Rattus norvegicus ILLUMINA 2019-12-24T00:00:00Z 199 adult pinealocytes adult siBsx Sprague-Dawley pineal gland siBsx
siBsx-3 SAMN13663583 SRX7427155 SRR10752353 SRS5873240 GSM4230127 SRP238459 PRJNA597111 NaN 6953 3130 RNA-Seq GEO public fastq,sra gs,ncbi,s3 Illumina HiSeq 2500 PAIRED cDNA TRANSCRIPTOMIC Rattus norvegicus ILLUMINA 2019-12-24T00:00:00Z 198 adult pinealocytes adult siBsx Sprague-Dawley pineal gland siBsx