Abstract: Disclosed are compositions, reagents, methods, kits and systems for improving un-fixing or decrosslinking of fixed biological particles, fixed membrane-bound particles, fixed cells, nuclei and/or the biomolecules (e.g., nucleic acids, RNA) of fixed cells or nuclei. Processing of nucleic acids and barcoding nucleic acids are disclosed. Methods for increasing amounts and quality of un-fixed biomolecules (e.g., nucleic acids, RNA) from fixed cells or nuclei are disclosed. Polymers like polyethylene glycol (PEG) increase the amount and quality of ribonucleic acid (RNA) obtained from un-fixed cells or nuclei. Disclosed are methods for using un-fixed cells/nuclei and biomolecules from un-fixed cells/nuclei in various assays. Polymers like PEG increase the efficiency of nucleic acid polymerase reactions (e.g., DNA polymerase, reverse transcriptase) in presence of un-fixing agents. In some examples, PEG increases reverse transcriptase activity, in the presence of proteases, when PEG is included in the reaction.