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