GSM5655848: biological replicate 3 of postnatal 5wks old mice; Mus musculus; RNA-Seq
Mice were sacrificed with an overdose of isoflurane inhalation followed by cervical dislocation. The entire colons were quickly removed before stripping the mucosal layers by sharp dissection. When the whole colon was opened to expose the mucosa along the mesenteric border, the proximal colon displayed a characteristic thick mucosal fold. This thick mucosal fold section was used for proximal preparation (1 or 2 cm below the ileocecal sphincter). For the distal colon, we used muscle preparations to 2 cm above the internal anal sphincter. These preparations were subsequently rinsed with Krebs-Ringer bicarbonate (Krebs) solution before being frozen in liquid nitrogen. Total RNA was isolated from proximal and distal colon tissues using the Direct-zol RNA Purification Kit (Zymo Research, Irvine, CA, USA) following which, the quality of total RNAs was determined using NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). Samples with an RNA integrity number (RIN) exceeding 8.5 were processed for preparation of libraries using a NEBNext Ultra II RNA Library Prep Kit from Illumina (Cat#E7770). RNA sequencing was performed using an Illumina NovaSeq 6000 platform at the DNA facility of Novogene Co. Ltd. (Beijing, China). Deep sequencing was conducted with 150 bp paired-end reads. Each biological replicate was considered as a separate sample.