title: GSM8244856: sci-mtChIL-seq,  embryo-21, MyoD-RNAPolII; Mus musculus; OTHER
library_construction_protocol: Trunks at E10.5 and E11.5 and limbs at E12.5, E13.5, and E14.5 were dissected from the embryos and cut into small pieces approximately 1 mm square and cell were isolated with protease treatment. Cell line were isolated with protease treatment. A total of 10-30k biotinylated cells per well were combined in 96-well avidin-coated plates and stained with primary antibody (1st Ab) and sci-ChIL probe. Tn5 transposition via Tn5 transposase integrates the sci-ChIL probe's indexed DNA into the genome around the primary antibody of the cell (cells are indexed with a unique sci-ChIL probe per well). The cells in the 96-well plate are then pooled, and 25 cells per well are added to another 96-well plate with a cell sorter, so that cells with different primary indexes are placed in the wells. The integrated regions are then amplified as RNA by in vitro transcription using T7 RNA polymerase. Transcribed RNA is purified and converted to cDNA for sequencing library preparation; a second index is added by PCR prior to sequencing. PCR is performed with a set of primers with unique i5 and i7 sequences per well. A library with a different primary index and the same secondary index was generated for each well. Overall, a library of approximately 2400 cells with different primary and secondary indices is produced. One single-target sequence includes one set of 96 sci-ChIL probes with T5 barcodes, and two sets of multi-target sequences is used.