title: GSM7100385: BLISS_CKp25_Step2_Replicate2; Mus musculus; OTHER
library_construction_protocol: BLISS was performed as previously described in Yan et al., 2017. Sorted neurons were attached to a MatTek 35 mm Dish | No. 1.0 Coverslip | 14 mm Glass Diameter | Poly-D-Lysine Coated (P35GC-1.0-14-C) by adding sorted cells to the center coverslip in 4% formaldehyde in PBS and spinning at 2000xg for 2 minutes. The attached nuclei were then end-repaired and A-tailed. Next BLISS adaptors were ligated overnight at 16°C. Excess BLISS adaptors were washed off and genomic DNA was isolated. Next DNA was sonicated to an average size of 500 bp and in-vitro transcribed. Transcribed RNA was isolated, and reverse transcribed to cDNA to prepare Illumina sequencing libraries. Custom BLISS adaptors containing RA3 and RA5 sequences were amplified using RP1 and  selected RPI Illumina primers as described in the Illumina TruSeq Small RNA Library Preparation Kit. Illumina libraries were sequenced in 75 bp single-end mode.